Anti-MCAM antibodies and associated methods of use

ABSTRACT

Described herein are anti-MCAM antibodies and antigen binding fragments thereof that are capable of inhibiting the interaction between MCAM and its ligand, a protein comprising a laminin α-4 chain. These anti-MCAM antibodies and antigen binding fragments thereof may be useful for, for example, treating inflammatory conditions characterized by the infiltration of MCAM-expressing cells into a site of inflammation in the body.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/021,777 filed Sep. 9, 2013, now U.S. Pat. No. 9,447,190, which claimspriority to U.S. Provisional Application No. 61/698,916, filed Sep. 10,2012, U.S. Provisional Application No. 61/797,179, filed Nov. 30, 2012,and U.S. Provisional Application No. 61/797,356, filed Dec. 15, 2012,each of the aforementioned applications is incorporated in its entiretyherein for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“481511_SEQLST.TXT,” created on Jul. 22, 2016 and containing 144,258bytes, which is incorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to antibodies that bind to melanomacell adhesion molecule (MCAM) which are capable of blocking theinteraction between MCAM and its ligand, the laminin alpha-4 chain. Thepresent invention is also directed to methods of use of the novelanti-MCAM antibodies described herein.

REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAMLISTING

The Sequence Listing written in file 436599SEQLIST.txt was created onSep. 16, 2013 for “ANTI-MCAM ANTIBODIES AND ASSOCIATED METHODS OF USE”is 144,190 bytes. The information contained in this file is herebyincorporated by reference.

BACKGROUND

A novel subset of CD4+ T cells, termed TH17 cells (T helper 17 cells),has been implicated in the pathogenesis of a number of autoimmunediseases, particularly those neuroinflammatory conditions involving CNSinfiltration of T cells, such as multiple sclerosis and the animalmodel, experimental autoimmune encephalomyelitis (EAE). See, e.g., Cuaet al., Nature 421: 744-748 (2003); see also Ivonov et al., Cell 126:1121-1133 (2006). Much attention on the enhanced pathogenicity of TH17cells has focused on their ability to secrete a number of selectcytokines including IL-17 and IL-22. However, the role of these TH17cytokines themselves has been called into question, as a conditionalknockout of IL-17 is insufficient to affect EAE progression. See, e.g.,Haak et al., J. Clin. Invest. 119: 61-69 (2009); see also Kreymborg etal., J. Immunol. 179: 8098-8104 (2007). Although IL-17 affects suchvital aspects of EAE as endothelial cell permeability, TH17 cells appearto do more than just produce any one cytokine. The moleculardeterminants of the pathogenic function of TH17 cells remain elusive.

The pathogenicity of TH17 cells can be partially explained by theirunique migration pattern as evidenced by their expression of chemokinereceptors. See, e.g., Kim, Inflamm. Allergy Drug Targets 8: 221-228(2009). It has been established that IL-17 producing cells are enrichedwithin the CCR6+ population of CD4+ T cells, likely conferring a uniquemigration pattern throughout the vasculature. See, e.g.,Acosta-Rodriguez et al., Nat. Immunol. 8:639-646 (2007). In fact, CCR6expression on T cells is required for T cell migration into the CNS andthe progression of EAE. Reboldi et al., Nat. Immunol. 10: 514-523(2009). A hypothesis has arisen of two waves of T cells, the first asmall population of CCR6 expressing TH17 cells that accumulates andrecruits a broader second wave of T cells with a more diverse chemokinereceptor repertoire. The anatomical site of this infiltration has beensuggested to be the choroid plexus due to the constitutive expression ofCCL20, a known ligand of CCR6. Ransohoff et al., Nat. Rev. Immunol. 3:569-581 (2003). The implication has been made that the true pathogenicfunction of TH17 cells lies in their specific recruitment andinfiltration of tissue.

Thus, there is still a need in the art to identify molecules that areinvolved in the infiltration of TH17 cells into CNS and contribute totheir pathogenicity. These molecules can be targets to designtherapeutic agents for neuroinflammatory conditions, such as multiplesclerosis (MS) and Parkinson's disease, as well as other TH17-mediatedinflammatory conditions not associated with the central nervous system.There is also a need to identify novel antibodies that can bind to andare capable of reducing, interfering, or otherwise blocking theinteraction between MCAM expressed on the surface of TH17 and itsidentified ligand.

SUMMARY OF THE INVENTION

TH17 cells play a significant role in the pathogenesis of variousautoimmune diseases, particularly those displaying neuroinflammatoryconditions involving T cells' infiltration into CNS. It has been newlydiscovered that (1) MCAM is selectively enriched on TH17 cells; and (2)MCAM interacts with a laminin α4 chain, such as, for example, the α4chain of laminin 411, present in the endothelial basement membrane. AnMCAM antagonist, e.g., a monoclonal antibody, capable of inhibitingMCAM's binding to a molecule containing a laminin α4 chain, such as, forexample, a laminin 411 molecule, may inhibit the migration of TH17 cellsinto CNS, and thus can be used as a therapeutic agent to prevent ortreat diseases displaying TH17-mediated neuroinflammatory conditions.MCAM antagonists, such as an MCAM monoclonal antibody or anantigen-binding fragment thereof, may also be useful to prevent or treatand TH17-mediated disease, including for example, autoimmune disease,for example, multiple sclerosis, inflammatory bowel disease, psoriasis,and rheumatoid arthritis.

The present invention is directed to novel antibodies that are capableof binding to MCAM protein on the surface of cells and, in turn, thatare capable of interfering with the interaction of MCAM with its ligand,a protein comprising a laminin alpha-4 chain. Optionally, the antibodyis a monoclonal antibody, antibody fragment, chimeric antibody,humanized antibody, single-chain antibody or antibody that competitivelyinhibits the binding of an anti-MCAM antibody to its respectiveantigenic epitope.

In other embodiments, the invention provides vectors comprising DNAencoding any of the herein described antibodies and host cellscomprising such vectors, wherein such host cells may be CHO cells, E.coli cells, or yeast cells. A process for producing any of the hereindescribed antibodies is further provided and comprises the steps ofculturing host cells under conditions suitable for expression of thedesired antibody, and recovering the desired antibody from the cellculture.

In one embodiment, the present invention is directed to an isolatedanti-MCAM antibody, or antigen binding fragment thereof, wherein theantibody or antigen binding fragment thereof comprises three light chainhypervariable regions (HVR-L1, HVR-L2, and HVR-L3) and three heavy chainhypervariable regions (HVR-H1, HVR-H2, and HVR-H3), and wherein:

(a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:31, HVR-L2comprises the amino acid sequence of SEQ ID NO:32, HVR-L3 comprises theamino acid sequence of SEQ ID NO:33, HVR-H1 comprises the amino acidsequence of SEQ ID NO:36, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:37, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:38;

(b) HVR-L1 comprises the amino acid sequence of SEQ ID NO:41, HVR-L2comprises the amino acid sequence of SEQ ID NO:42, HVR-L3 comprises theamino acid sequence of SEQ ID NO:43, HVR-H1 comprises the amino acidsequence of SEQ ID NO:46, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:47, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:48;

(c) HVR-L1 comprises the amino acid sequence of SEQ ID NO:51, HVR-L2comprises the amino acid sequence of SEQ ID NO:52, HVR-L3 comprises theamino acid sequence of SEQ ID NO:53, HVR-H1 comprises the amino acidsequence of SEQ ID NO:56, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:57, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:58;

(d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:61, HVR-L2comprises the amino acid sequence of SEQ ID NO:62, HVR-L3 comprises theamino acid sequence of SEQ ID NO:63, HVR-H1 comprises the amino acidsequence of SEQ ID NO:66, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:67, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:68;

(e) HVR-L1 comprises the amino acid sequence of SEQ ID NO:73, HVR-L2comprises the amino acid sequence of SEQ ID NO:74, HVR-L3 comprises theamino acid sequence of SEQ ID NO:75, HVR-H1 comprises the amino acidsequence of SEQ ID NO:78, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:79, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:80; or

(f) HVR-L1 comprises the amino acid sequence of SEQ ID NO:85, HVR-L2comprises the amino acid sequence of SEQ ID NO:86, HVR-L3 comprises theamino acid sequence of SEQ ID NO:87, HVR-H1 comprises the amino acidsequence of SEQ ID NO:90, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:91, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:92.

In certain embodiments, the anti-MCAM antibody may be a chimeric orhumanized antibody. In another embodiment, the anti-MCAM antibody may bean IgG1 antibody which may optionally be produced in bacteria or CHOcells.

In yet another embodiment, the present invention is directed to anisolated anti-MCAM antibody, or antigen binding fragment thereof, saidantibody or antigen binding fragment thereof comprising a light chainvariable region and a heavy chain variable region, wherein:

(a) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:30 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:35;

(b) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:40 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:45;

(c) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:50 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:55;

(d) the light chain variable region comprises the amino acid sequence ofSEQ ID NO:60 and the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:65;

(e) the light chain variable region comprises the amino acid sequence ofany one of SEQ ID NOS:70, 71, or 72 and the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:77; or

(f) the light chain variable region comprises the amino acid sequence ofany one of SEQ ID NOS:83 or 84 and the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO:89.

In certain embodiments, the anti-MCAM antibody may be a chimeric orhumanized antibody. In another embodiment, the anti-MCAM antibody may bean IgG1 antibody which may optionally be produced in bacteria or CHOcells.

In yet another embodiment, the present invention is directed to anisolated anti-MCAM antibody, or antigen binding fragment thereof, thatbinds substantially the same epitope as, or competes for binding with,any of the anti-MCAM antibodies described herein.

In yet other embodiments, the present invention is directed to anisolated anti-MCAM antibody, or antigen binding fragment thereof, thatblocks the interaction between an MCAM protein comprising the amino acidsequence of SEQ ID NO:22 and a protein comprising a laminin α-4 chain.Another embodiment of the present invention is directed to an isolatedanti-MCAM antibody, or antigen binding fragment thereof, that blocks theinteraction between an MCAM protein comprising the amino acid sequencesof SEQ ID NOS:22 and 23 and a protein comprising a laminin α-4 chain. Afurther embodiment of the present invention is directed to an isolatedanti-MCAM antibody or antigen binding fragment thereof which does notblock the interaction between an MCAM protein consisting of the aminoacid sequence of SEQ ID NO:22 and a protein comprising a laminin α-4chain. Yet another embodiment of the present invention is directed to anisolated anti-MCAM antibody, or antigen binding fragment thereof, thatblocks the interaction between an MCAM protein comprising the amino acidsequences of SEQ ID NOS:22, 23, and 24 and a protein comprising alaminin α-4 chain. Further embodiments of the present invention aredirected to isolated anti-MCAM antibodies, or antigen binding fragmentsthereof, that bind to antigenic epitopes defined by domains 1 and 2, ordomain 3 of the human MCAM protein. In a preferred embodiment, theanti-MCAM antibody or fragment thereof does not bind to a proteinconsisting of amino acids 19-129 of the human MCAM protein.

Yet other embodiments of the present invention are directed topharmaceutical compositions comprising any of the herein describedantibodies, or antigen binding fragment thereof, and articles ofmanufacture comprising the same.

Other embodiments of the present invention are directed to the use of ananti-MCAM antibody, or antigen binding fragment thereof, in themanufacture of a medicament for the treatment of an inflammatorydisorder characterized by infiltration of MCAM-expressing cells into asite of inflammation in the body. In certain embodiments, theinflammatory disorder may be a central nervous system (CNS) inflammatorydisorder characterized by infiltration of MCAM-expressing cells into theCNS.

The invention also provides for the use of an anti-MCAM antibody, orantigen binding fragment thereof, in the manufacture of a medicament forthe treatment of multiple sclerosis, Parkinson's disease. The inventionalso provides for the use of an anti-MCAM antibody, or antigen bindingfragment thereof, in the manufacture of a medicament for the treatmentof allergic contact dermatitis. The invention also provides for the useof an anti-MCAM antibody, or antigen binding fragment thereof, in themanufacture of a medicament for the treatment of, psoriasis. Theinvention also provides for the use of an anti-MCAM antibody, or antigenbinding fragment thereof, in the manufacture of a medicament for thetreatment of psoriatic arthritis. The invention also provides for theuse of an anti-MCAM antibody, or antigen binding fragment thereof, inthe manufacture of a medicament for the treatment of cancer, forexample, a solid tumor, such as a melanoma. The invention also providesfor the use of an anti-MCAM antibody, or antigen binding fragmentthereof, in the manufacture of a medicament for the treatment ofsarcoidosis.

Another embodiment of the present invention is directed to a method forthe treatment of an inflammatory disorder characterized by infiltrationof MCAM-expressing cells to a site of inflammation, the methodcomprising administering to a mammalian subject in need thereof aneffective amount of an anti-MCAM antibody or antigen binding fragmentthereof that inhibits the binding of MCAM to a protein comprising alaminin α-4 chain. In certain embodiments, the mammalian subject may bea human and the MCAM-expressing cells may be TH17 cells.

In another aspect, the present invention provides an isolated h1749anti-MCAM antibody, or antigen binding fragment thereof. In oneembodiment, the antibody or antigen binding fragment thereof comprisesthree light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) andthree heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3),wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 61,HVR-L2 comprises the amino acid sequence of SEQ ID NO:62, HVR-L3comprises the amino acid sequence of SEQ ID NO:63, HVR-H1 comprises theamino acid sequence of SEQ ID NO:66, HVR-H2 comprises the amino acidsequence of SEQ ID NO:67, HVR-H3 comprises the amino acid sequence ofSEQ ID NO:68. In another embodiment, the isolated anti-MCAM antibody, orantibody binding fragment thereof, further comprises a heavy chainframework region 2 (FR2) comprising the amino acid sequence of SEQ IDNO: 128.

In one other aspect, the present invention provides an isolated h2107anti-MCAM antibody, or antigen binding fragment thereof. In oneembodiment, the isolated anti-MCAM antibody, or antigen binding fragmentthereof, comprises three light chain hypervariable regions (HVR-L1,HVR-L2, and HVR-L3) and three heavy chain hypervariable regions (HVR-H1,HVR-H2, and HVR-H3), wherein HVR-L1 comprises the amino acid sequence ofSEQ ID NO:85, HVR-L2 comprises the amino acid sequence of SEQ ID NO:86,HVR-L3 comprises the amino acid sequence of SEQ ID NO:87, HVR-H1comprises the amino acid sequence of SEQ ID NO:90, HVR-H2 comprises theamino acid sequence of SEQ ID NO:91, and HVR-H3 comprises the amino acidsequence of SEQ ID NO:92. In another embodiment, the isolated anti-MCAMantibody, or antibody binding fragment thereof, further comprises aheavy chain framework region 2 (FR2) comprising the amino acid sequenceof SEQ ID NO:134 and/or a heavy chain framework region 3 (FR3)comprising the amino acid sequence of SEQ ID NO:137. In one otherembodiment, the isolated anti-MCAM antibody, or antibody bindingfragment thereof, further comprises a light chain framework region 2(FR2) comprising the amino acid sequence of SEQ ID NO:146; and/or alight chain framework region 3 (FR3) comprising the amino acid sequenceof SEQ ID NO:144.

In one additional aspect, the present invention provides an isolatedh2120 anti-MCAM antibody, or antigen binding fragment thereof. In oneembodiment, the antibody or antigen binding fragment thereof comprisesthree light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) andthree heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3),wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO:73, HVR-L2comprises the amino acid sequence of SEQ ID NO:74, HVR-L3 comprises theamino acid sequence of SEQ ID NO:75, HVR-H1 comprises the amino acidsequence of SEQ ID NO:141, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:79, and HVR-H3 comprises the amino acid sequence of SEQ IDNO:80. In another embodiment, the isolated anti-MCAM antibody, orantibody binding fragment thereof, comprises a heavy chain frameworkregion 2 (FR2) comprising the amino acid sequence of SEQ ID NO:134and/or a heavy chain framework region 3 (FR3) comprising the amino acidsequence of SEQ ID NO:135. In one other embodiment, the isolatedanti-MCAM antibody, or antibody binding fragment thereof, furthercomprises a light chain framework region 1 (FR1) comprising the aminoacid sequence of SEQ ID NO:147; a light chain framework region 2 (FR2)comprising the amino acid sequence of SEQ ID NO:148; a light chainframework region 3 (FR3) comprising the amino acid sequence of SEQ IDNO:149; or any combination thereof.

Additional embodiments of the present invention will be evident to thoseof ordinary skill in the art based upon the teachings of the presentspecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. FIGS. 1A and 1B depict the presence of MCAM inIL-17-producing human CD4+ cells. FIG. 1A depicts the microarrayanalysis showing that MCAM is an up-regulated gene in both circulatingand activated TH17 cells. FIG. 1B depicts the cell sorting resultsshowing that MCAM exist almost exclusively in a small population ofmemory T cells (CD45RO+ T cells). FIG. 1C depicts the cell sortingresults showing that MCAM is enriched in IL-17-producing human CD4+ Tcells.

FIGS. 2A, B depict the surface markers of MCAM expressing T cells. FIG.2A depicts MCAM expressing T cells as effector memory T cells (CCR6+while CCR7−). FIG. 2B depicts the integrin expression pattern of MCAMexpressing T cells. The majority of MCAM expressing T cells are integrinα4 positive, but are largely integrin β7 negative and β1 positive.

FIGS. 3A-F depict the effects of various cytokines on CD4+/CD45RO+memory T cells. FIG. 3A depicts the effects of various cytokines onIL-17 production in MCAM positive T cells. FIG. 3B depicts thepercentage of cells expressing MCAM following stimulation by variouscytokines. FIGS. 3C, 3D, and 3E depict the levels of IL-17 (FIG. 3C),IL-22 (FIG. 3D), and CCL20 (FIG. 3E) in both MCAM positive and MCAMnegative cells after stimulations with various cytokines. FIG. 3Fdepicts the intracellular levels of FOXP3 in both MCAM positive and MCAMnegative cells after stimulations with various cytokines.

FIGS. 4A-H depict the identification of laminin 411 as the MCAM ligand.FIG. 4A depicts co-localization of the MCAM ligand and laminin on thechoroid plexus of healthy mice. FIG. 4B depicts absence of MCAM stainingon the choroid plexus of healthy mice (4′,6-diamidino-2-phenylindole(DAPI) was used as a counterstain). FIG. 4C depicts the presence of MCAMon vascular endothelial cells within healthy mouse brain (DAPI was usedas a counterstain). FIG. 4D depicts the expression pattern of the MCAMligand by staining healthy mouse spinal cord sections with MCAM-Fcprotein. FIG. 4E depicts co-localization of the MCAM ligand and lamininon healthy mouse spinal cord. FIG. 4F depicts the extracellular matrix(ECM) localization of the MCAM ligand. CD31 staining was used to showthat MCAM staining is exterior to the endothelial cell layer within thevasculature. FIG. 4G depicts the localization of the MCAM ligand withinEAE lesions. MCAM-Fc is shown to colocalize with laminin within theendothelial cell basement membrane, but not within the parenchymalbasement membrane. FIG. 4H depicts co-localization of the MCAM ligandand laminin 411 (or laminin alpha-4 chain).

FIGS. 5A-C. FIG. 5A depicts specific binding of MCAM antibodies to humanand mouse MCAM. FIG. 5B depicts blockage of MCAM-Fc's binding to tissuesby MCAM antibodies. FIG. 5C depicts inhibition of the interactionbetween human MCAM and its ligand laminin 411 by a monoclonal antibody.

FIGS. 6A, B. FIG. 6A depicts the light chain variable region of clone 17monoclonal antibody. FIG. 6A discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:1) and the aminoacid sequence of the light chain variable region (SEQ ID NO:2), in orderof appearance. The three hypervariable regions are also indicated asCDRL1 (SEQ ID NO:3), CDRL2 (SEQ ID NO:4), and CDRL3 (SEQ ID NO:5). FIG.6B depicts the heavy chain variable region clone 17 monoclonal antibody.FIG. 6B discloses the nucleic acid sequence encoding the heavy chainvariable region (SEQ ID NO:6) and the amino acid sequence of the heavychain variable region (SEQ ID NO:7), in order of appearance. The threehypervariable regions are also indicated as CDRH1 (SEQ ID NO:8), CDRH2(SEQ ID NO:9), and CDRH3 (SEQ ID NO:10).

FIGS. 7A, B. FIG. 7A depicts absence of MCAM on T cells from naivemouse. FIG. 7B depicts MCAM expression levels among splenocytes in thepresence of various cytokines. Splenocytes were obtained from PLPimmunized SJL mice and in vitro restimulated with PLP.

FIGS. 8A, B depicts the effects of MCAM blockade on disease progressionin a therapeutic model of EAE. After EAE symptoms appeared,PLP-immunized mice were treated intraperitoneally with (1) anti-MCAMantibody (clone 15) at 10 mg/kg body weight, (2) the isotype control(Bioxcell) at 10 mg/kg body weight, and (3) PBS every day thereafter.The disease progression (FIG. 8A) and body weights (FIG. 8B) weremonitored every 2-3 days. Data represent the mean of 15 mice±sem(standard error of the mean).

FIGS. 9A, B. FIG. 9A depicts the light chain variable region of clone 15monoclonal antibody. FIG. 9A discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:12) and the aminoacid sequence of the light chain variable region (SEQ ID NO:13), inorder of appearance. The three hypervariable regions are also indicatedas CDRL1 (SEQ ID NO:14), CDRL2 (SEQ ID NO:15), and CDRL3 (SEQ ID NO:16).FIG. 9B depicts the heavy chain variable region clone 15 monoclonalantibody. FIG. 9B discloses the nucleic acid sequence encoding the heavychain variable region (SEQ ID NO:17) and the amino acid sequence of theheavy chain variable region (SEQ ID NO:18), in order of appearance. Thethree hypervariable regions are also indicated as CDRH1 (SEQ ID NO:19),CDRH2 (SEQ ID NO:20), and CDRH3 (SEQ ID NO:21).

FIGS. 10A, B depict the results of a domain binding test for MCAMantibodies.

FIGS. 11A, B depict the amino acid sequence (A) (SEQ ID NO:11—AccessionNo. CAA48332) and structure (B) for human MCAM. In FIG. 11A, the aminoacid residue positions corresponding to the five immunoglobulin domainsof human MCAM are as follows—1: amino acid residues 19-129; 2: aminoacid residues 139-242; 3: amino acid residues 244-321; 4: amino acidresidues 335-424; and 5: amino acid residues 430-510) (SEQ IDNOS:22-26), which are also depicted schematically in FIG. 11B.

FIGS. 12A, B show the amino acid sequences for two α4-chain isoforms ofhuman laminin 411. FIG. 12A shows the amino acid sequence correspondingto GenBank Accession No. NP001098676 (SEQ ID NO:27) and FIG. 12B showsthe amino acid sequence corresponding to GenBank Accession No.NP001098677 (SEQ ID NO:28).

FIG. 13 depicts the light chain variable region of clone 1174.1.3monoclonal antibody. FIG. 13 discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:29) and the aminoacid sequence of the light chain variable region (SEQ ID NO:30), inorder of appearance. The three hypervariable regions are also indicatedas CDRL1 (SEQ ID NO:31), CDRL2 (SEQ ID NO:32), and CDRL3 (SEQ ID NO:33).

FIG. 14 depicts the heavy chain variable region clone 1174.1.3monoclonal antibody. FIG. 14 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:34) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:35), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:36), CDRH2 (SEQ ID NO:37), and CDRH3 (SEQ ID NO:38).

FIG. 15 depicts the light chain variable region of clone 1414.1.2monoclonal antibody. FIG. 15 discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:39) and the aminoacid sequence of the light chain variable region (SEQ ID NO:40), inorder of appearance. The three hypervariable regions are also indicatedas CDRL1 (SEQ ID NO:41), CDRL2 (SEQ ID NO:42), and CDRL3 (SEQ ID NO:43).

FIG. 16 depicts the heavy chain variable region clone 1414.1.2monoclonal antibody. FIG. 16 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:44) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:45), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:46), CDRH2 (SEQ ID NO:47), and CDRH3 (SEQ ID NO:48).

FIG. 17 depicts the light chain variable region of clone 1415.1.1monoclonal antibody. FIG. 17 discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:49) and the aminoacid sequence of the light chain variable region (SEQ ID NO:50), inorder of appearance. The three hypervariable regions are also indicatedas CDRL1 (SEQ ID NO:51), CDRL2 (SEQ ID NO:52), and CDRL3 (SEQ ID NO:53).

FIG. 18 depicts the heavy chain variable region clone 1415.1.1monoclonal antibody. FIG. 18 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:54) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:55), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:56), CDRH2 (SEQ ID NO:57), and CDRH3 (SEQ ID NO:58).

FIG. 19 depicts the light chain variable region of clone 1749.1.3monoclonal antibody. FIG. 19 discloses the nucleic acid sequenceencoding the light chain variable region (SEQ ID NO:59) and the aminoacid sequence of the light chain variable region (SEQ ID NO:60), inorder of appearance. The three hypervariable regions are also indicatedas CDRL1 (SEQ ID NO:61), CDRL2 (SEQ ID NO:62), and CDRL3 (SEQ ID NO:63).

FIG. 20 depicts the heavy chain variable region clone 1749.1.3monoclonal antibody. FIG. 20 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:64) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:65), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:66), CDRH2 (SEQ ID NO:67), and CDRH3 (SEQ ID NO:68).

FIGS. 21A, B depict different versions of the light chain variableregion of clone 2120.4.19 monoclonal antibody. FIG. 21A-B discloses oneversion of the nucleic acid sequence encoding a light chain variableregion (SEQ ID NO:69), the amino acid sequence of version 1 of the lightchain variable region (SEQ ID NO:70), the amino acid sequence of version2 of the light chain variable region (SEQ ID NO:71), and the amino acidsequence of version 3 of the light chain variable region (SEQ ID NO:72).The three hypervariable regions are also indicated as CDRL1 (SEQ IDNO:73), CDRL2 (SEQ ID NO:74), and CDRL3 (SEQ ID NO:75).

FIG. 22 depicts the heavy chain variable region clone 2120.4.19monoclonal antibody. FIG. 22 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:76) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:77), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:78), CDRH2 (SEQ ID NO:79), and CDRH3 (SEQ ID NO:80).

FIGS. 23A, B depict different versions of the light chain variableregion of clone 2107.4.10 monoclonal antibody. FIGS. 23A-B discloses thenucleic acid sequence encoding version 1 of a light chain variableregion (SEQ ID NO:81), the nucleic acid sequence encoding version 2 of alight chain variable region (SEQ ID NO:83), the amino acid sequence ofversion 1 of the light chain variable region (SEQ ID NO:82), and theamino acid sequence of version 2 of the light chain variable region (SEQID NO:84). The three hypervariable regions are also indicated as CDRL1(SEQ ID NO:85), CDRL2 (SEQ ID NO:86), and CDRL3 (SEQ ID NO:87).

FIG. 24 depicts the heavy chain variable region clone 2107.4.10monoclonal antibody. FIG. 24 discloses the nucleic acid sequenceencoding the heavy chain variable region (SEQ ID NO:88) and the aminoacid sequence of the heavy chain variable region (SEQ ID NO:89), inorder of appearance. The three hypervariable regions are also indicatedas CDRH1 (SEQ ID NO:90), CDRH2 (SEQ ID NO:91), and CDRH3 (SEQ ID NO:92).

FIG. 25A shows the alignment of sequences of the variable heavy chainsfor the following: murine 1749.1.3 anti-MCAM antibody (1749.1.3_VH_pro;SEQ ID NO:93); 1749 VH1 humanized anti-MCAM antibody (h1749VH1; SEQ IDNO:94); 1749 VH2 humanized anti-MCAM antibody (h1749VH2; SEQ ID NO:95);and heavy chain human variable IGHV3-7*02 sequence used as the frameworkdonor (U96282_VH; SEQ ID NO:96). Kabat numbering is used andhypervariable regions (HVRs) grafted from the murine 1749.1.3 antibodyto the variable heavy chain variable IGHV3-7*02 framework are boxed. Thebolded amino acid residues in the humanized antibody sequences differfrom the corresponding residues in the murine antibody sequence. Theposition of canonical and interface amino acid residues that may affectCDR contact or CDR structure are indicated by an asterisk.

FIG. 25B shows the alignment of sequences of the variable light chainsfor the following: murine 1749.1.3 anti-MCAM antibody (1749.1.3_VL_pro;SEQ ID NO:97); 1749 VL1 humanized anti-MCAM antibody (h1749VL1 SEQ IDNO:98); 1749 VL2 humanized anti-MCAM antibody (h1749VL2 SEQ ID NO:99);and light chain human variable X02990 IGKV4-1*01 sequence used as theframework donor (X02990_VL SEQ ID NO:100). Kabat numbering is used andhypervariable regions (HVRs) grafted from the murine 1749.1.3 antibodyto the variable light chain variable X02990 IGKV4-1*01 framework areboxed. The bolded amino acid residues in the humanized antibodysequences differ from the corresponding residues in the murine antibodysequence. The position of canonical and interface amino acid residuesthat may affect CDR contact or CDR structure are indicated by anasterisk.

FIG. 26A shows the alignment of sequences of the variable heavy chainsfor the following: murine 2107.4.10.18 anti-MCAM antibody(2107.4.10.18_VH_topo_pro; SEQ ID NO:101); 2107 VH1 humanized anti-MCAMantibody (h2107VH1; SEQ ID NO:102); 2107 VH2 humanized anti-MCAMantibody (h2107VH2; SEQ ID NO:103); 2107 VH3 humanized anti-MCAMantibody (h2107VH3; SEQ ID NO:104); 2107 VH4 humanized anti-MCAMantibody (h2107VH4; SEQ ID NO:105); 2107 VH5 humanized anti-MCAMantibody (h2107VH5; SEQ ID NO:106); 2107 VH6 humanized anti-MCAMantibody (h2107VH6; SEQ ID NO:107); and heavy chain human variableAF062133 IGHV2-26*01 sequence used as the framework donor (AF062133_VH;SEQ ID NO:108). Kabat numbering is used and hypervariable regions (HVRs)grafted from the murine 2107.4.10.18 antibody to the variable heavychain variable AF062133 IGHV2-26*01 framework are boxed in both FIG.26A. The S30T, I37V, L48I and K71R mutations combined with (i) mutationsof the boxed N/D residues between CDR-H2 and CDR-H3 (D78N) restoresmurine N-glycosylation; or a mutation in an N-G sequence in CDR-H1,e.g., N32S (VH4); N32Q (VH5); or G33A (VH6)), provides an N deamidationmutant. The bolded amino acid residues in the humanized antibodysequences differ from the corresponding residues in the murine antibodysequence. The position of canonical and interface amino acid residuesthat may affect CDR contact or CDR structure are indicated by anasterisk.

FIG. 26B shows the alignment of sequences of the variable light chainsfor the following: murine 2107_L7-6 anti-MCAM antibody (2107_L7-6_pro;SEQ ID NO:109); 2107 VL1 humanized anti-MCAM antibody (h2107VL1; SEQ IDNO:110); 2107 VL2 humanized anti-MCAM antibody (h2107VL2; SEQ IDNO:111); 2107 VL3 humanized anti-MCAM antibody (h2107VL3 SEQ ID NO:112);and light chain human variable U86803 IGKV1-27*01 sequence used as theframework donor (U86803_VL SEQ ID NO:113). Kabat numbering is used andhypervariable regions (HVRs) grafted from the murine 2107_L7-6 antibodyto the variable light chain variable U86803 IGKV1-27*01 framework areboxed. The bolded amino acid residues in the humanized antibodysequences differ from the corresponding residues in the murine antibodysequence. The position of canonical and interface amino acid residuesthat may affect CDR contact or CDR structure are indicated by anasterisk.

FIG. 27A shows the alignment of sequences of the variable heavy chainsfor the following: murine 2120.4.19.6 anti-MCAM antibody(2120.4.19.6_VH_topo_pro; SEQ ID NO:114); 2120 VH1 humanized anti-MCAMantibody (h2120VH1; SEQ ID NO:115); 2120 VH2 humanized anti-MCAMantibody (h2120VH2; SEQ ID NO:116); 2120 VH3 humanized anti-MCAMantibody (h2120VH3; SEQ ID NO:117); 2120 VH4 humanized anti-MCAMantibody (h2120VH4; SEQ ID NO:118); 2120 VH5 humanized anti-MCAMantibody (h2120VH5; SEQ ID NO:119); and heavy chain human variableAF062133 IGHV2-26*01 sequence used as the framework donor (AF062133_VH;SEQ ID NO:108). Kabat numbering is used and hypervariable regions (HVRs)grafted from the murine 2120.4.19.6 antibody to the variable heavy chainvariable AF062133 IGHV2-26*01 framework are boxed. The S30T, I37V, L48Iand K71R mutations combined with (i) mutations of the boxed N/D residuesin CDR-H1, e.g., N32S (VH3); N32Q (VH4); or G33A (VH5)), provides an Ndeamidation mutant. The bolded amino acid residues in the humanizedantibody sequences differ from the corresponding residues in the murineantibody sequence. The position of canonical and interface amino acidresidues that may affect CDR contact or CDR structure are indicated byan asterisk. Residues where mutations were focused due to the presenceof N-deamination sites or N-glycosylation sites are shown in thebracketed box.

FIG. 27B shows the alignment of sequences of the variable light chainsfor the following: murine 2120.4.19.6 anti-MCAM antibody(2120.4.19.6_VL_topo_pro; SEQ ID NO:120); 2120 VL1 humanized anti-MCAMantibody (h2120VL1 SEQ ID NO:121); 2120 VL2 humanized anti-MCAM antibody(h2120VL2 SEQ ID NO:122); 2120 VL3 humanized anti-MCAM antibody(h2120VL3 SEQ ID NO:123); and light chain human variable X84343IGKV2-26*01 sequence used as the framework donor (X84343_VL SEQ IDNO:124). Kabat numbering is used and hypervariable regions (HVRs)grafted from the murine 2120.4.19.6 antibody to the variable light chainvariable X84343 IGKV2-26*01 framework are boxed. The bolded amino acidresidues in the humanized antibody sequences differ from thecorresponding residues in the murine antibody sequence. The position ofcanonical and interface amino acid residues that may affect CDR contactor CDR structure are indicated by an asterisk.

FIG. 28A-C compare the blocking of various 1749, 2120, and 2107antibodies respectively of MCAM binding to laminin 411.

FIGS. 29A, B shows the % inhibition for certain humanized anti-MCAMantibodies as compared to chimeric anti-MCAM antibodies.

FIG. 30: Treatment with an anti-MCAM antibody reduces inflammation inmodel of skin inflammation.

FIGS. 31A, B: Anti-MCAM antibodies inhibit melanoma growth by volume (A)and weight (B) in xenograft model.

DETAILED DESCRIPTION

1. Definitions and Abbreviations

1.1. Definitions

An “individual” or “subject” as used herein may be any of mammaliananimals (e.g., domesticated animals), including human, dog, cat, cattle,horse, goat, pig, swine, sheep, monkey, guinea pig, rat, and mouse. Inone embodiment, the individual or subject can be a human.

“MCAM” (melanoma cell adhesion molecule, also known as CD146 and MUC18)refers to a cell surface glycoprotein belonging to the immunoglobulinsuperfamily involved in cell adhesion, and in cohesion of theendothelial monolayer at intercellular junctions in vascular tissue. Italso promotes tumor progression of many cancers, such as solid tumors,including melanoma and prostate cancer. It is known to interact in ahomotypic/homophilic manner and may also bind to other ligands. Thehuman MCAM has the amino acid sequence of SEQ ID NO: 11 (FIG. 11A),which includes five immunoglobulin domains (1: amino acid residues19-129; 2: amino acid residues 139-242; 3: amino acid residues 244-321;4: amino acid residues 335-424; and 5: amino acid residues 430-510)shown as SEQ ID NOS:22-26, which are also depicted schematically in FIG.11B.

A “laminin α4 chain” refers to one of the polypeptide chains found inlaminin molecules, which are expressed in the basal lamina (of thebasement membrane), a protein network foundation for most cells andorgans. Laminins are known to bind to cell membranes through plasmamembrane molecules and contribute to cell attachment. The laminin α4chain typically forms a complex with a laminin β-chain, and a lamininγ-chain. The laminin α4 chain is found in numerous laminin moleculesincluding, without limitation, laminin 411 (laminin 8 or α4β1γ1);laminin 421 (laminin 9 or α4β2γ1), and laminin 423 (laminin 14 orα4β2γ3). There are two main isoforms of the human laminin α4-chain:GenBank Accession Nos. NP001098676 and NP001098677 as shown in FIGS.12A-B (amino acid sequences SEQ ID NOS:27-28). “Laminin 411” refers to atrimeric polypeptide complex made up of three polypeptide subunits orchains: α4-chain, a β1-chain, and a γ1-chain.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes aqualitative biological activity of an MCAM polypeptide. For the purposeof the present invention, the biological activity preferably is theability to inhibit the ability of MCAM (i) to specifically bind itsligand: a laminin α4 chain, e.g., the α4 chain of laminin 411; and/or(ii) to facilitate an MCAM-expressing cell, e.g., a TH17 cell, toinfiltrate into or migrate to a subject's tissue. Antagonists of MCAMcan be identified, for example, based upon their ability to inhibit orblock the specific binding of MCAM to its ligand: a laminin α4 chain,e.g., the α4 chain of laminin 411. MCAM antagonists specificallyinclude, without limitation, antibodies (e.g., antagonist orneutralizing antibodies), including chimeric, humanized and humanantibodies and their functional fragments, small molecules, ribozymes,aptamers, peptides, and nucleic acids that encode polypeptideantagonists or antagonist antibodies.

The term “MCAM antagonist antibody” refers to an antibody which inhibitsor neutralizes the activity of MCAM. Such an antibody specifically bindsto a polypeptide target involved in the infiltration of anMCAM-expressing cell into the CNS, e.g., MCAM or a laminin α4 chain(e.g., the α4 chain of laminin 411).

A “blocking” antibody, “neutralizing” antibody, or “antagonist” antibodyis one which inhibits or reduces a biological activity of the antigen itbinds. Such antibodies may substantially or completely inhibit thebiological activity of the antigen.

The terms “specifically binds” or “binds specifically” as used hereinmeans that one member of a specific binding pair will not show anystatistically significant binding to molecules other than its specificbinding partner. A binding partner may show at least 1000 times theaffinity of binding (measured as an apparent association constant) forits specific binding pair partner than a non-specific binding partner.For example, antibodies that bind to MCAM with a binding affinity of 10⁷mole/L or more, typically 10⁸ mole/L or more, are said to bindspecifically to MCAM.

The terms “biological activity” and “biologically active” with regard toMCAM refer to its ability to specifically bind its ligand (a laminin α4chain, e.g., the α4 chain of laminin 411) and/or to facilitate theinfiltration of MCAM-expressing cells, e.g., TH17 cells, into the CNS.

The term an “MCAM-expressing cell” refers to a cell of the immune systemthat expresses MCAM. For example, MCAM expression is enriched on memoryT lymphocytes, e.g., TH17 cells.

The term “binding molecule” as used herein refers to a molecule thatspecifically binds to a target. The term specifically includes, withoutlimitation, antibodies and antibody fragments (e.g. those comprising oneor more of the CDRs described herein), and peptide and non-peptide smallmolecules.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingsome common structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind can be, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The term “antibody” used herein may encompass intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. The term “antigen-binding fragment” of an antibody refers to aportion of the full-length immunoglobulin molecule that specificallybinds to the antigen. An antigen-binding fragment of an antibody thusincludes an antigen-binding heavy chain, light chain, heavy chain-lightchain dimer, Fab fragment, F(ab′)₂ fragment, Fv fragment, single chainFv (scFv), diabodies, linear antibodies, and multispecific antibodiesformed from antibody fragment(s).

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are substantiallysimilar and bind the same epitope(s), except for possible variants thatmay arise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. Such monoclonal antibodytypically includes an antibody comprising a variable region that binds atarget, wherein the antibody was obtained by a process that includes theselection of the antibody from a plurality of antibodies. For example,the selection process can be the selection of a unique clone from aplurality of clones, such as a pool of hybridoma clones, phage clones orrecombinant DNA clones. It should be understood that the selectedantibody can be further altered, for example, to improve affinity forthe target, to humanize the antibody, to improve its production in cellculture, to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered variableregion sequence is also a monoclonal antibody of this invention. Inaddition to their specificity, the monoclonal antibody preparations areadvantageous in that they are typically uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including thehybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies(see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al.,J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol.338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093(2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004);and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004) andtechnologies for producing human or human-like antibodies from animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893,WO/9634096, WO/9633735, and WO/91 10741, Jakobovits et al., Proc. Natl.Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Pat. Nos.5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.5,545,807; WO 97/17852, U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio/Technology,10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994);Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93(1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences, as well as “humanized” antibodies.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

An “intact antibody” herein is one which comprises two antigen bindingregions, and an Fc region. Preferably, the intact antibody has afunctional Fc region.

An “antibody (or any other binding molecule) that binds to the sameepitope” as a reference antibody (or any other binding molecule) refersto an antibody (or any other binding molecule) that blocks binding ofthe reference antibody (or any other binding molecule) to its antigen ina competition assay by 50% or more, and conversely, the referenceantibody (or any other binding molecule) blocks binding of the antibodyto its antigen in a competition assay by 50% or more.

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J.Mol. Biol. 226:889-896 (1992).

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called κ and λ, based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains, intactantibodies can be assigned to different “classes.” There are five majorclasses of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and severalof these may be further divided into “subclasses” (isotypes), e.g.,IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domainsthat correspond to the different classes of antibodies are called α, δ,ε, γ, and μ, respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Hypervariable region” or “HVR” refers to the amino acid residues of anantibody that are responsible for antigen-binding. The hypervariableregion generally comprises amino acid residues from a “complementaritydetermining region” or “CDR” (Kabat et al., Sequences of Proteins ofImmunological Interest, 5^(th) Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (Chothia and Lesk, J. Mol. Biol. 196: 901-917(1987)).

The term “complementarity determining regions” or “CDRs” when usedherein refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity. The CDRs ofimmunological receptors are the most variable part of the receptorprotein, giving receptors their diversity, and are carried on six loopsat the distal end of the receptor's variable domains, three loops comingfrom each of the two variable domains of the receptor.

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens. Typically, an epitope refersto a unit of structure conventionally bound by an immunoglobulin VH-VLpair. Epitopes define the minimum binding site for an antibody, and thusrepresent the target of specificity of an antibody. Epitopes can belinear or conformational, and can be as small as three amino acids.

A “small molecule” is defined herein to have a molecular weight belowabout 600, preferably below about 1000 daltons. Generally, a smallmolecule is a non-peptide small organic molecule.

“Isolated,” when used to describe the various polypeptides, proteins andantibodies disclosed herein, means polypeptide, protein or antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials that would typically interfere with diagnosticor therapeutic uses for the polypeptide, protein or antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide, protein or antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide, protein or antibody includespolypeptide, protein or antibody in situ within recombinant cells, sinceat least one component of the associated natural environment will not bepresent. Ordinarily, however, isolated polypeptide, protein or antibodywill be prepared by at least one purification step.

The terms “affinity”, “binding affinity” and “K_(d)” refer to theequilibrium dissociation constant (expressed in units of concentration)associated with each MCAM binding molecule—target complex, such asbetween an anti-MCAM antibody and MCAM. The binding affinity is directlyrelated to the ratio of the off-rate constant (generally reported inunits of inverse time, e.g., seconds⁻¹) to the on-rate constant(generally reported in units of concentration per unit time, e.g.,molar/second). The binding affinity may be determined by, for example,an ELISA assay, kinetic exclusion assay or surface plasmon resonance. Itis noted that certain epitopes can occur repetitively (multivalent) on acell surface and that the dissociation constant (koff) for the bindingof an antibody to a repetitive epitope may be greatly diminished overthe dissociation constant for the reaction of the same antibody with thecorresponding ligand in univalent form. The diminished dissociationconstant arises because when one antibody-ligand bond dissociates, otherbonds hold the bivalent (or multivalent) antibody to the multivalentligand, allowing the dissociated bond to form again. The dissociationconstant for the reaction between bivalent (or multivalent) Ab andmultivalent ligand has been termed the functional affinity to contrastit with intrinsic affinity, which is the association constant for anantibodies representative individual site.

The terms “dissociation”, “dissociation rate” and “k_(off)” as usedherein, are intended to refer to the off rate constant for dissociationof a binding molecule, such as an antibody, from the bindingmolecule/target, e.g. antibody/antigen complex.

The terms “association”, “association rate” and “k_(on)” as used herein,are intended to refer to the on rate constant for association of abinding molecule with a target, such as an antibody with an antigen, toform a complex.

The terms “effective concentration” and “EC₅₀” as used herein, areintended to refer to the concentration of a binding molecule (e/g/antibody) capable of interacting with sufficient quantities of targetmolecules to produce an effect on approximately 50% of the treatedcells.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis/prevention, or during the course ofclinical pathology. The term refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain thedesired effect for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic ortherapeutic result. An effective amount refers to the amount of activecompound or pharmaceutical agent that elicits the biological ormedicinal response in a tissue, system, animal, individual or human thatis being sought by a researcher, veterinarian, medical doctor or otherclinician, which includes one or more of the following:

(A) preventing the disease; for example, preventing an inflammatorydisease, such as a neuroinflammatory disease, condition or disorder inan individual that may be predisposed to the disease, condition ordisorder but does not yet experience or display the pathology orsymptoms of the disease,

(B) inhibiting the disease; for example, inhibiting an inflammatorydisease, such as a neuroinflammatory disease, condition or disorder inan individual that is experiencing or displaying the pathology orsymptoms of the disease, condition or disorder (i.e., arresting furtherdevelopment of the pathology and/or symptoms), and

(C) ameliorating the disease; for example, ameliorating an inflammatorydisease, such as a neuroinflammatory disease, condition or disorder inan individual that is experiencing or displaying the pathology orsymptoms of the disease, condition or disorder (i.e., reversing thepathology and/or symptoms).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. Before the presentmethods, kits and uses therefore are described, it is to be understoodthat this invention is not limited to the particular methodology,protocols, cell lines, animal species or genera, constructs, andreagents described as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein, the singular forms “a”, “and”, and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an antibody” includes aplurality of such antibodies and reference to “the dosage” includesreference to one or more dosages and equivalents thereof known to thoseskilled in the art, and so forth. Throughout this specification andclaims, the word “comprise,” or variations such as “comprises” or“comprising,” will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

1.2. Abbreviations

Abs antibodies CDR complementarity determining region CFA completeFreund's adjuvant CFSE carboxyfluorescein succinimidyl ester CNS centralnervous system DAPI 4′,6-diamidino-2-phenylindole DN dopamine-containingneuron EAE experimental autoimmune encephalomyelitis ECM extracellularmatrix FACS fluorescence Activated cell sorting FR Framework Region IFAincomplete Freund's adjuvant Igs immunoglobulins MCAM melanoma celladhesion molecule MOG myelin oligodendrocyte glycoprotein (MOG) MSmultiple sclerosis PD Parkinson's disease PMA phorbol myristate acetate2. MCAM

MCAM (melanoma cell adhesion molecule) is a cell-surface glycoproteinoriginally identified as a melanoma antigen, whose expression isassociated with tumor progression and the development of metastaticpotential. MCAM is a 113 kDa cell surface integral membrane glycoproteincomposed of a signal peptide, five immunoglobulin-like domains (1, 2, 3,4, and 5; or V-V-C2-C2-C2), a transmembrane region, and a shortcytoplasmic tail. See, e.g., Lehmann et al., Proc. Nat'l Acad. Sci. USA86: 9891-9895 (1989) and FIG. 11B. MCAM is a member of theimmunoglobulin superfamily and has significant sequence homology to anumber of cell adhesion molecules of the Ig superfamily, including BEN(Pourquie et al., Proc. Nat'l Acad. Sci. USA 89: 5261-5265 (1992)),neural-cell adhesion molecule (N-CAM) (Owens et al., Proc. Nat'l Acad.Sci. USA 84: 294-298 (1987)), myelin-associated glycoprotein (MAG) (Laiet al., Proc. Nat'l Acad. Sci. USA 84: 4337-4341 (1987)), deleted incolorectal cancer protein (DCC) (Hedrick et al., Genes Devel. 8:1174-1183 (1994)), and gicerin (Taira et al., Neuron 12: 861-872(1994)). The expression of MCAM has been detected in relatively limitedspectrum of normal human tissues and in a variety of malignantneoplasms. In normal adult tissues, MCAM is expressed on endothelialcells, smooth muscle cells (Shih et al., Lab. Invest. 75: 377-388(1996); Sers et al., Cancer Res. 54: 5689-5694 (1994)), a subpopulationof activated T lymphocytes (Pickl et al., J. Immunol. 158: 2107-2115(1997)), and intermediate trophoblasts (Shih et al., supra). MCAM isalso expressed on a variety of malignant neoplasms including smoothmuscle neoplasms (Leiomyomas and leiomyosarcomas), tumors of vascularorigin (angiosarcomas and Kaposi's sarcomas), placental sitetrophoblastic tumors, choriocarcinomas, and melanomas (Shih et al.,Clinical Cancer Res. 2: 569-575 (1996); Holzmann et al., Int. J. Cancer39: 466-471 (1987)). The expression of MUC18 correlates directly withthe metastatic potential of human melanoma cells (Bar-Eli, CancerMetastasis, 18: 377-385 (1999)).

A number of studies have identified MCAM as a marker of tumorprogression and metastasis in melanomas. The expression of MCAM isabsent in normal melanocytes and benign nevi but prominent on manyprimary melanomas and in most metastatic lesions (Lehmann et al., supra;Shih et al., supra). MCAM expression correlates well with tumor verticalthickness and metastasis formation, and greater than 80% of metastaticlesions express MCAM (Lehmann et al., supra; Xie et al., Cancer Res. 57:2295-2303 (1997); and Shih et al., supra). Modulators of MCAM have beengenerated to treat melanomas. See, e.g., U.S. Pat. No. 7,067,131.Recently, MCAM modulation has been suggested to identify and selectinflammatory cytokine-secreting T cells or their precursors to treatvarious inflammatory conditions. See, e.g., U.S. Published PatentApplication No. 2011/0014183.

3. Neuroinflammatory Conditions, Multiple Sclerosis, and ParkinsonDisease

A neuroinflammatory condition refers to a condition associated withinflammation of the nervous system, in an embodiment the central nervoussystem (CNS), and which is associated with cell/tissue damage. It istypically characterized by, for example, increased glial activation,increased pro-inflammatory cytokine/chemokine levels (e.g., TNFα, INFγ,IL-1β), increased blood-brain-barrier permeability, and/or increasedimmune cell (e.g., leukocyte) recruitment/invasion to the CNS. It mayrefer to, for example, chronic neuroinflammation, such as aninflammation associated with chronic activation of cells of the immunesystem (i.e., autoimmune-associated neuroinflammation). Such chronicneuroinflammation can be observed in, for example, multiple sclerosis(MS). Additionally, Parkinson's disease (PD) is a neurodegenerativedisease displaying neuroinflammation, for example, activated microgliaand infiltrating T cells.

Multiple sclerosis, as a progressive neurological autoimmune disease,results from chronic, pathological inflammation (Yednock et al., Nature356: 63-66 (1992); Baron et al., J. Exp. Med. 177: 57-68 (1993)). MSaffects an estimated 250,000 to 350,000 people in the United States.Multiple sclerosis is thought to be the result of a specific autoimmunereaction wherein certain leukocytes attack and initiate the destructionof myelin, the insulating sheath covering nerve fibers. The onset of MSmay be dramatic or so mild as to not cause a patient to seek medicalattention. The most common symptoms include weakness in one or morelimbs, visual blurring due to optic neuritis, sensory disturbances,diplopia, and ataxia. The course of disease may be stratified into threegeneral categories: (1) relapsing MS, (2) chronic progressive MS, and(3) inactive MS.

Relapsing MS is generally characterized by recurrent attacks ofneurologic dysfunction. MS attacks generally evolve over days to weeksand may be followed by complete, partial, or no recovery. Recovery fromattacks generally occurs within weeks to several months from the peak ofsymptoms, although rarely some recovery may continue for 2 or moreyears.

Chronic progressive MS results in gradually progressive worseningwithout periods of stabilization or remission. This form develops inpatients with a prior history of relapsing MS, although in 20% ofpatients, no relapses can be recalled. Acute relapses also may occurduring the progressive course of MS.

A third form is inactive MS. Inactive MS is characterized by fixedneurologic deficits of variable magnitude. Most patients with inactiveMS have an earlier history of relapsing MS. The course of MS is alsodependent on the age of the patient. For example, favorable prognosticfactors include early onset (excluding childhood), a relapsing courseand little residual disability 5 years after onset. By contrast, poorprognosis is associated with a late age of onset (i.e., age 40 or older)and a progressive course. These variables are interdependent, sincechronic progressive MS tends to begin at a later age that relapsing MS.Disability from chronic progressive MS is usually due to progressiveparaplegia or quadriplegia in individual patients.

Parkinson's disease (PD) is a progressive neurodegenerative diseasedisplaying primary clinical features of motor abnormalities, e.g.,resting tremor, bradykinesia, and rigidity. PD is characterized by theloss of dopamine-containing neuron (DN) cells in the substantia nigraparts compacta (Forno, J. Neurophthol. Exp. Neurol. 55: 259-272 (1996)).One of the hallmarks of PD is neuroinflammation characterized byactivated microglia and infiltrating T cells. Although studies havesuggested various mechanisms for PD, such as mitochonodrial dysfunction,oxidative stress, and impairment of protein degradation machinery, thecause of PD remains elusive (Dauer et al., Neuron 39: 889-909 (2003)).Recent findings have indicated that both innate and adaptive immunitymay play important roles in the pathogenesis of PD (Stone et al.,Antioxid. Redox. Signal. 11: 2151-2166 (2009)). Particularly, it hasbeen shown in the animal model of PD that both activated microglia and Tlymphocytes contribute significantly to neurodegeneration. See, e.g.,Brochard et al., J. Clin. Invest. 119: 182-192 (2009). It has beenhypothesized that CD4 positive T cells (e.g., proinflammatory T17 cells)mediate cytotoxicity by activating microglia in PD and/or exert a directtoxic effect on substantia nigra DNs (Appel, J. Clin. Invest. 119: 13-15(2009)).

4. Autoimmune Diseases

An autoimmune disease herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis,acute immunological arthritis, chronic inflammatory arthritis,degenerative arthritis, type II collagen-induced arthritis, infectiousarthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis,Still's disease, vertebral arthritis, and juvenile-onset rheumatoidarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis, andankylosing spondylitis), inflammatory hyperproliferative skin diseases,psoriasis such as plaque psoriasis, gutatte psoriasis, pustularpsoriasis, and psoriasis of the nails, atopy including atopic diseasessuch as hay fever and Job's syndrome, dermatitis including contactdermatitis, chronic contact dermatitis, exfoliative dermatitis, allergicdermatitis, allergic contact dermatitis, dermatitis herpetiformis,nummular dermatitis, seborrheic dermatitis, non-specific dermatitis,primary irritant contact dermatitis, and atopic dermatitis, x-linkedhyper IgM syndrome, allergic intraocular inflammatory diseases,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria, myositis,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO),inflammatory bowel disease (IBD) (for example, Crohn's disease,autoimmune-mediated gastrointestinal diseases, colitis such asulcerative colitis, colitis ulcerosa, microscopic colitis, collagenouscolitis, colitis polyposa, necrotizing enterocolitis, and transmuralcolitis, and autoimmune inflammatory bowel disease), bowel inflammation,pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,respiratory distress syndrome, including adult or acute respiratorydistress syndrome (ARDS), meningitis, inflammation of all or part of theuvea, iritis, choroiditis, an autoimmune hematological disorder,rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema,cranial nerve damage as in meningitis, herpes gestationis, pemphigoidgestationis, pruritis scroti, autoimmune premature ovarian failure,sudden hearing loss due to an autoimmune condition, IgE-mediateddiseases such as anaphylaxis and allergic and atopic rhinitis,encephalitis such as Rasmussen's encephalitis and limbic and/orbrainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,proliferative nephritis, autoimmune polyglandular endocrine failure,balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, allergic reaction, eczema including allergic or atopiceczema, asteatotic eczema, dyshidrotic eczema, and vesicularpalmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma,and autoimmune asthma, conditions involving infiltration of T cells andchronic inflammatory responses, immune reactions against foreignantigens such as fetal A-B-O blood groups during pregnancy, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, lupus, including lupus nephritis, lupus cerebritis,pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus anddiscoid lupus erythematosus, alopecia lupus, systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), adult onset diabetesmellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetesinsipidus, diabetic retinopathy, diabetic nephropathy, diabeticlarge-artery disorder, immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytes,tuberculosis, sarcoidosis, granulomatosis including lymphomatoidgranulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides,including vasculitis, large-vessel vasculitis (including polymyalgiarheumatica and giant-cell (Takayasu's) arteritis), medium-vesselvasculitis (including Kawasaki's disease and polyarteritisnodosa/periarteritis nodosa), microscopic polyarteritis,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS) and ANCA-associated small-vesselvasculitis, temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), pernicious anemia (anemia perniciosa), Addison's disease, purered cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, CNS inflammatory disorders, multiple organ injurysyndrome such as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behçet's disease/syndrome, Castleman's syndrome, Goodpasture'ssyndrome, Reynaud's syndrome, Sjögren's syndrome, Stevens-Johnsonsyndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid,pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, thermal injury,preeclampsia, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,thrombocytopenia (as developed by myocardial infarction patients, forexample), including thrombotic thrombocytopenic purpura (TTP),post-transfusion purpura (PTP), heparin-induced thrombocytopenia, andautoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, polyglandular syndromes such asautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis(LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrésyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, acute febrile neutrophilicdermatosis, subcorneal pustular dermatosis, transient acantholyticdermatosis, cirrhosis such as primary biliary cirrhosis andpneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliacdisease, celiac sprue (gluten enteropathy), refractory sprue, idiopathicsprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; LouGehrig's disease), coronary artery disease, autoimmune ear disease suchas autoimmune inner ear disease (AIED), autoimmune hearing loss,polychondritis such as refractory or relapsed or relapsingpolychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal or segmental or focal segmentalglomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,chorioretinitis, autoimmune hepatological disorder, fibromyalgia,multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastricatrophy, presenile dementia, demyelinating diseases such as autoimmunedemyelinating diseases and chronic inflammatory demyelinatingpolyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyly, and telangiectasia), male and femaleautoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, parasitic diseases suchas leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathicpulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatumet diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, flariasis, cyclitis such as chroniccyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, SCID, acquired immune deficiency syndrome (AIDS),echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemickeratoconjunctivitis, idiopathic nephritic syndrome, minimal changenephropathy, benign familial and ischemia-reperfusion injury, transplantorgan reperfusion, retinal autoimmunity, joint inflammation, bronchitis,chronic obstructive airway/pulmonary disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, sympatheticophthalmia, rheumatic diseases, mixed connective tissue disease,nephrotic syndrome, insulitis, polyendocrine failure, autoimmunepolyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism(AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisisbullosa acquisita (EBA), hemochromatosis, myocarditis, nephroticsyndrome, primary sclerosing cholangitis, purulent or nonpurulentsinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, orsphenoid sinusitis, an eosinophil-related disorder such as eosinophilia,pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonaryeosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, seronegativespondyloarthritides, polyendocrine autoimmune disease, sclerosingcholangitis, sclera, episclera, chronic mucocutaneous candidiasis,Bruton's syndrome, transient hypogammaglobulinemia of infancy,Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis,autoimmune disorders associated with collagen disease, rheumatism,neurological disease, lymphadenitis, reduction in blood pressureresponse, vascular dysfunction, tissue injury, cardiovascular ischemia,hyperalgesia, renal ischemia, cerebral ischemia, and diseaseaccompanying vascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, ischemic re-perfusiondisorder, reperfusion injury of myocardial or other tissues,lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses withacute inflammatory components, multiple organ failure, bullous diseases,renal cortical necrosis, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

5. Cancer

Cancer or a cancerous condition is the physiological condition inmammals that is typically characterized by unregulated cellgrowth/proliferation. Included in this definition are benign andmalignant cancers, as well metastatic cancers. Also included are solidtumors and hematopoietic malignancies. Metastatic cancer refers to acancer that has spread from the place where it first started to anotherplace in the body. Tumors formed by metastatic cancer cells are called ametastatic tumor or a metastasis (which is also used to refer to theprocess by which cancer cells spread to other parts of the body). Ingeneral, metastatic cancer has the same name and same type of cancercells as the original, or primary, cancer. Metastatic cancer includesprostate cancer, lung cancer, and pancreas cancer. By prostate cancer orconditions related to prostate cancer is meant the malignant growth ofabnormal cells in the prostate gland, capable of invading and destroyingother prostate cells, and spreading (metastasizing) to other parts ofthe body, including bones, lungs, liver, and lymph nodes. By lung canceror conditions related to lung cancer is meant the malignant growth ofabnormal cells in the lungs, capable of invading and destroying otherlung cells, and spreading (metastasizing) to other parts of the body,including the adrenal gland, and liver. By pancreatic cancer orconditions related to pancreatic cancer is meant the malignant growth ofabnormal cells in the pancreas, capable of invading and destroying otherpancreas cells, and spreading (metastasizing) to other parts of thebody, including the liver, lungs, and peritoneum.

6. MCAM Antagonists

The present invention provides antagonists of MCAM. Such antagonistsencompass those that directly act upon MCAM (e.g., an anti-MCAMantibody) and those that indirectly affect MCAM activity (e.g., ananti-laminin α4 chain antibody). Such antagonists are useful, forexample, for treating a central nervous system (CNS) inflammatorydisorder characterized by infiltration of MCAM-expressing cells into theCNS. In one embodiment, a composition comprising an MCAM antagonist isuseful for reducing inflammation in a mammalian subject. In anotherembodiment, such a composition is useful for partially or fullyinhibiting CNS infiltration of MCAM-expressing cells. Examples of MCAMantagonists include, without limitation, antagonist or neutralizingantibodies or antibody fragments against one or more domains, e.g., animmunoglobulin domain of a native sequence MCAM polypeptide or a domainof a native sequence laminin α4 chain polypeptide (e.g., the α4 chain oflaminin 411), small molecules, ribozymes, aptamers, peptides, andnucleic acids that encode polypeptide antagonists or antagonistantibodies. Reference to “an” antagonist encompasses a singleantagonist. In one embodiment, the MCAM antagonists are antibodiesincluding, without limitation, chimeric, humanized and human antibodiesand their functional fragments.

In a preferred embodiment, the laminin α4 chain is an α4 chain oflaminin 411. In another preferred embodiment, the MCAM antagonist blocksthe interaction of an MCAM domain comprising the amino acid sequence ofSEQ ID NO:22 and/or SEQ ID NO:23 with a laminin α4 chain.

6.1 Screening Assays to Identify MCAM Antagonists

The present invention includes screening assays to identify MCAMantagonists, which find utility in the treatment of inflammatoryconditions characterized by infiltration of MCAM-expressing cells intothe central nervous system (CNS).

In one aspect, the invention concerns a method for identifying aninhibitor of CNS infiltration by MCAM-expressing cells comprising thesteps of: (a) incubating a population of cells expressing a laminin α4chain, e.g., an α4 chain of laminin 411, with MCAM, in the presence orabsence of a candidate molecule; (b) monitoring the level of binding ofMCAM to the cells; and (c) identifying said candidate molecule as aninhibitor of CNS infiltration by MCAM-expressing cells if the level ofMCAM binding is lower in the presence than in the absence of saidcandidate molecule. In one embodiment, the candidate molecule isselected from the group consisting of a small molecule, a peptide, apolypeptide, and an antibody. Those of ordinary skill in the art willappreciate that other types of candidate molecule may be suitable. Inanother embodiment, the level of binding of MCAM is monitored by knowntechniques including, without limitation, fluorescent microscopy, FACS,and ELISA. In one other embodiment, the cells expressing a laminin α4chain are endothelial cells. In a preferred embodiment, the laminin α4chain is an α4 chain of laminin 411.

Screening assays for antagonist drug candidates may be designed toidentify compounds that bind or complex with MCAM (including a subunitor other fragment thereof) or with an MCAM ligand, such as a laminin α4chain (e.g., an α4 chain of laminin 411), or otherwise interfere withthe interaction of MCAM with other cellular proteins, therebyinterfering with the interaction of MCAM with its ligand, e.g., alaminin α4 chain. The screening assays provided herein include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Generally, binding assays and activity assays are provided.

The assays can be performed in a variety of formats, including, withoutlimitation, protein-protein binding assays, biochemical screeningassays, immunoassays, and cell-based assays, which are wellcharacterized in the art.

All assays for antagonists and agonists are common in that they call forcontacting the drug candidate with an MCAM polypeptide, or an MCAMligand polypeptide, e.g., a laminin α4 chain, or a fragment of suchpolypeptides (specifically including MCAM and laminin α4 chains) underconditions and for a time sufficient to allow these two components tointeract.

For example, human MCAM is a 646 amino acid polypeptide, the sequence ofwhich is available from the GenBank database under Accession NumberAAA20922.1 (CAA48332) (SEQ ID NO:11; FIG. 11A). Amino acid sequences forhuman laminin α4-chain are available from the GenBank database underAccession Nos. NP001098676 and NP001098677 (SEQ ID NOS: 27-28; FIGS.12A-B). The making of antibodies or small molecules binding to suchpolypeptides is well within the skill of the ordinary artisan.

In binding assays, the interaction is binding, and the complex formedcan be isolated or detected in the reaction mixture. In a particularembodiment, either the MCAM or MCAM ligand polypeptide or the drugcandidate is immobilized on a solid phase, e.g., on a microtiter plate,by covalent or non-covalent attachments. Non-covalent attachmentgenerally is accomplished by coating the solid surface with a solutionof the MCAM or MCAM ligand polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the MCAMor MCAM ligand polypeptide to be immobilized can be used to anchor it toa solid surface. The assay is performed by adding the non-immobilizedcomponent, which may be labeled by a detectable label, to theimmobilized component, e.g., the coated surface containing the anchoredcomponent. When the reaction is complete, the non-reacted components areremoved, e.g., by washing, and complexes anchored on the solid surfaceare detected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound is a polypeptide which interacts with but doesnot bind to MCAM or the MCAM ligand polypeptide, its interaction withthe respective polypeptide can be assayed by methods well known fordetecting protein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fieldsand Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991).

Compounds that interfere with the interaction of MCAM and otherextracellular components, in particular an MCAM ligand polypeptide, canbe tested as follows. Usually a reaction mixture is prepared containingMCAM and the extracellular component (e.g., MCAM ligand such as alaminin α4 chain, e.g., an α4 chain of laminin 411) under conditions andfor a time allowing for the interaction of the two products. To test theability of a candidate compound to inhibit the interaction of MCAM andits ligand, the reaction is run in the absence and in the presence ofthe test compound. In addition, a placebo may be added to a thirdreaction mixture, to serve as positive control. Since MCAM has beenshown to specifically bind its ligand, e.g., a laminin α4 chain, theability of the test compound to inhibit the MCAM/MCAM ligand interactioncan, for example, be tested by measuring the degree of binding betweenMCAM and its ligand in the absence and presence of the test compound. Ifthe degree of MCAM binding to its ligand is lower in the absence of thecandidate compound than in its presence, the candidate compound is anMCAM antagonist by the definition of the present invention.

An alternate screening protocol involves the use of a population ofcells expressing a laminin α4 chain, e.g., an α4 chain of laminin 411,which can be incubated with MCAM, in the presence and absence of a testcompound, and binding of MCAM to the cell population monitored, e.g. byfluorescent microscopy (exemplified in Example 5). Other methods ofmonitoring will be appreciated by those skilled in the art, includingfluorescence-activated cell sorting (FACS) and enzyme-linkedimmunosorbent assay (ELISA). If the binding of MCAM to the cellpopulation in the presence of the test compound is lower than in itsabsence, the test compound is an MCAM antagonist.

The MCAM antagonists identified based upon their ability to inhibit thebinding of MCAM to its ligand, e.g., a laminin α4 chain, are drugcandidates for the treatment of neuroinflammatory conditionscharacterized by infiltration of MCAM-expressing cells into the CNS.

It is emphasized that the screening assays specifically discussed hereinare for illustration only. A variety of other assays, which can beselected depending on the type of the antagonist candidates screened(e.g. polypeptides, peptides, non-peptide small organic molecules,aptamers, ribozymes, nucleic acid, etc.) are well known to those skilledin the art and are equally suitable for the purposes of the presentinvention.

6.2 Antibodies

In one aspect, an MCAM antagonist is an anti-MCAM antibody or ananti-laminin α4 chain, e.g., α4 chain of laminin 411, antibody, or anantigen-binding fragment thereof. In some embodiments, an anti-MCAMantibody is a blocking antibody that fully or partially blocks theinteraction of MCAM with its ligand, a laminin α4 chain. In otherembodiments, an anti-laminin α4 chain antibody is a blocking antibodythat fully or partially blocks the interaction of a laminin α4 chainwith MCAM. In certain embodiments, the anti-MCAM antibody binds to theextracellular domain of MCAM which interacts with its ligand, a lamininα4 chain. In a preferred embodiment, the laminin α4 chain is an α4 chainof laminin 411.

In one embodiment, an anti-MCAM antibody specifically or selectivelybinds to an MCAM fragment comprising or having the amino acid sequenceof position 19 to position 129 of SEQ ID NO: 11 (SEQ ID NO:22). Inanother embodiment, an anti-MCAM antibody specifically or selectivelybinds to an MCAM fragment comprising or having the amino acid sequenceof position 139 to position 242 of SEQ ID NO: 11 (SEQ ID NO:23). In oneother embodiment, an anti-MCAM antibody specifically or selectivelybinds to an MCAM fragment comprising the amino acid sequences of SEQ IDNOS:22 and 23.

In a preferred embodiment, the antagonist antibody blocks theinteraction of an MCAM domain comprising the amino acid sequence of SEQID NO:22 and/or SEQ ID NO:23 with a laminin α4 chain.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:3;    -   b) HVR-L2 shown as SEQ ID NO:4;    -   c) HVR-L3 shown as SEQ ID NO:5;    -   d) HVR-H1 shown as SEQ ID NO:8;    -   e) HVR-H2 shown as SEQ ID NO:9; and/or    -   f) HVR-H3 shown as SEQ ID NO:10.

In another embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:2 and/or aheavy chain variable domain shown as SEQ ID NO:7.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:14;    -   b) HVR-L2 shown as SEQ ID NO:15;    -   c) HVR-L3 shown as SEQ ID NO:16;    -   d) HVR-H1 shown as SEQ ID NO:19;    -   e) HVR-H2 shown as SEQ ID NO:20; and/or    -   f) HVR-H3 shown as SEQ ID NO:21.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:13 and/or aheavy chain variable domain shown as SEQ ID NO:18.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:31;    -   b) HVR-L2 shown as SEQ ID NO:32;    -   c) HVR-L3 shown as SEQ ID NO:33;    -   d) HVR-H1 shown as SEQ ID NO:36;    -   e) HVR-H2 shown as SEQ ID NO:37; and/or    -   f) HVR-H3 shown as SEQ ID NO:38.

In another embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:30 and/or aheavy chain variable domain shown as SEQ ID NO:35.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:41;    -   b) HVR-L2 shown as SEQ ID NO:42;    -   c) HVR-L3 shown as SEQ ID NO:43;    -   d) HVR-H1 shown as SEQ ID NO:46;    -   e) HVR-H2 shown as SEQ ID NO:47; and/or    -   f) HVR-H3 shown as SEQ ID NO:48.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:40 and/or aheavy chain variable domain shown as SEQ ID NO:45.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:51;    -   b) HVR-L2 shown as SEQ ID NO:52;    -   c) HVR-L3 shown as SEQ ID NO:53;    -   d) HVR-H1 shown as SEQ ID NO:56;    -   e) HVR-H2 shown as SEQ ID NO:57; and/or    -   f) HVR-H3 shown as SEQ ID NO:58.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:50 and/or aheavy chain variable domain shown as SEQ ID NO:55.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:61;    -   b) HVR-L2 shown as SEQ ID NO:62;    -   c) HVR-L3 shown as SEQ ID NO:63;    -   d) HVR-H1 shown as SEQ ID NO:66;    -   e) HVR-H2 shown as SEQ ID NO:67; and/or    -   f) HVR-H3 shown as SEQ ID NO:68.

In another embodiment, the anti-MCAM antibody or antibody fragmentfurther comprises a heavy chain framework region 2 (FR2) comprising theamino acid sequence of SEQ ID NO: 128.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as SEQ ID NO:60 and/or aheavy chain variable domain shown as SEQ ID NO:65.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:73;    -   b) HVR-L2 shown as SEQ ID NO:74;    -   c) HVR-L3 shown as SEQ ID NO:75;    -   d) HVR-H1 shown as SEQ ID NO:78;    -   e) HVR-H2 shown as SEQ ID NO:79; and/or    -   f) HVR-H3 shown as SEQ ID NO:80.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as any one of SEQ IDNOS:70, 71, or 72 and/or a heavy chain variable domain shown as SEQ IDNO:77.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:85;    -   b) HVR-L2 shown as SEQ ID NO:86;    -   c) HVR-L3 shown as SEQ ID NO:87;    -   d) HVR-H1 shown as SEQ ID NO:90;    -   e) HVR-H2 shown as SEQ ID NO:91; and/or    -   f) HVR-H3 shown as SEQ ID NO:92.

In another embodiment, the anti-MCAM antibody or antibody fragmentfurther comprises (a) a heavy chain framework region 2 (FR2) comprisingthe amino acid sequence of SEQ ID NO: 134; (b) a heavy chain frameworkregion 3 (FR3) comprising the amino acid sequence of SEQ ID NO:137; (c)a light chain framework region 2 (FR2) comprising the amino acidsequence of SEQ ID NO:146; and/or (d) a light chain framework region 3(FR3) comprising the amino acid sequence of SEQ ID NO:144.

In one other embodiment, the anti-MCAM antibody or antibody fragmentcomprises a light chain variable domain shown as any one of SEQ IDNOS:83 or 84 and/or a heavy chain variable domain shown as SEQ ID NO:89.

In other embodiments, the anti-MCAM antibody or antibody fragmentcomprises the following hypervariable regions (HVRs):

-   -   a) HVR-L1 shown as SEQ ID NO:73;    -   b) HVR-L2 shown as SEQ ID NO:74;    -   c) HVR-L3 shown as SEQ ID NO:75;    -   d) HVR-H1 shown as SEQ ID NO:141;    -   e) HVR-H2 shown as SEQ ID NO:79; and/or    -   f) HVR-H3 shown as SEQ ID NO:80.

In another embodiment, the anti-MCAM antibody or antibody fragmentfurther comprises (a) a heavy chain framework region 2 (FR2) comprisingthe amino acid sequence of SEQ ID NO: 134; (b) a heavy chain frameworkregion 3 (FR3) comprising the amino acid sequence of SEQ ID NO:135; (c)a light chain framework region 1 (FR1) comprising the amino acidsequence of SEQ ID NO:147; (d) a light chain framework region 2 (FR2)comprising the amino acid sequence of SEQ ID NO:148; and/or (e) a lightchain framework region 3 (FR3) comprising the amino acid sequence of SEQID NO:149.

In another aspect, the present invention provides MCAM antagonists thatbind to substantially the same epitope as, or compete for binding with,an anti-MCAM antibody described herein. In one embodiment, the MCAMantagonist binds to substantially the same epitope as, or competes forbinding with, an anti-MCAM antibody comprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:3;    -   b) HVR-L2 shown as SEQ ID NO:4;    -   c) HVR-L3 shown as SEQ ID NO:5;    -   d) HVR-H1 shown as SEQ ID NO:8;    -   e) HVR-H2 shown as SEQ ID NO:9; and/or    -   f) HVR-H3 shown as SEQ ID NO:10.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:2 and/or aheavy chain variable domain shown as SEQ ID NO:7.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:14;    -   b) HVR-L2 shown as SEQ ID NO:15;    -   c) HVR-L3 shown as SEQ ID NO:16;    -   d) HVR-H1 shown as SEQ ID NO:19;    -   e) HVR-H2 shown as SEQ ID NO:20; and/or    -   f) HVR-H3 shown as SEQ ID NO:21.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:13 and/or aheavy chain variable domain shown as SEQ ID NO:18.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:31;    -   b) HVR-L2 shown as SEQ ID NO:32;    -   c) HVR-L3 shown as SEQ ID NO:33;    -   d) HVR-H1 shown as SEQ ID NO:36;    -   e) HVR-H2 shown as SEQ ID NO:37; and/or    -   f) HVR-H3 shown as SEQ ID NO:38.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:30 and/or aheavy chain variable domain shown as SEQ ID NO:35.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:41;    -   b) HVR-L2 shown as SEQ ID NO:42;    -   c) HVR-L3 shown as SEQ ID NO:43;    -   d) HVR-H1 shown as SEQ ID NO:46;    -   e) HVR-H2 shown as SEQ ID NO:47; and/or    -   f) HVR-H3 shown as SEQ ID NO:48.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:40 and/or aheavy chain variable domain shown as SEQ ID NO:45.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:51;    -   b) HVR-L2 shown as SEQ ID NO:52;    -   c) HVR-L3 shown as SEQ ID NO:53;    -   d) HVR-H1 shown as SEQ ID NO:56;    -   e) HVR-H2 shown as SEQ ID NO:57; and/or    -   f) HVR-H3 shown as SEQ ID NO:58.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:50 and/or aheavy chain variable domain shown as SEQ ID NO:55.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:61;    -   b) HVR-L2 shown as SEQ ID NO:62;    -   c) HVR-L3 shown as SEQ ID NO:63;    -   d) HVR-H1 shown as SEQ ID NO:66;    -   e) HVR-H2 shown as SEQ ID NO:67; and/or    -   f) HVR-H3 shown as SEQ ID NO:68.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as SEQ ID NO:60 and/or aheavy chain variable domain shown as SEQ ID NO:65.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:73;    -   b) HVR-L2 shown as SEQ ID NO:74;    -   c) HVR-L3 shown as SEQ ID NO:75;    -   d) HVR-H1 shown as SEQ ID NO:78;    -   e) HVR-H2 shown as SEQ ID NO:79; and/or    -   f) HVR-H3 shown as SEQ ID NO:80.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as any one of SEQ IDNOS:70, 71, or 72 and/or a heavy chain variable domain shown as SEQ IDNO:77.

In one other embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising the following HVRs:

-   -   a) HVR-L1 shown as SEQ ID NO:85;    -   b) HVR-L2 shown as SEQ ID NO:86;    -   c) HVR-L3 shown as SEQ ID NO:87;    -   d) HVR-H1 shown as SEQ ID NO:90;    -   e) HVR-H2 shown as SEQ ID NO:91; and/or    -   f) HVR-H3 shown as SEQ ID NO:92.

In another embodiment, the MCAM antagonist binds to substantially thesame epitope as, or competes for binding with, an anti-MCAM antibodycomprising a light chain variable domain shown as any one of SEQ IDNOS:83 or 84 and/or a heavy chain variable domain shown as SEQ ID NO:89.

In an embodiment, the invention provides humanized antibodies. In someembodiments, the humanized antibody comprises a heavy chain variabledomain sequence selected from the group consisting of SEQ ID NO: 94 andSEQ ID NO: 95 (FIG. 25A; h1749). In some embodiments, the humanizedantibody comprises a light chain variable domain sequence selected fromthe group consisting of SEQ ID NO: 98 and SEQ ID NO: 99 (FIG. 25B;h1749). In another embodiment, the humanized antibody comprises a heavychain variable domain sequence SEQ ID NO: 94 and a light chain variabledomain sequence selected from the group consisting of SEQ ID NO: 98 andSEQ ID NO:99. In another embodiment, the humanized antibody comprises aheavy chain variable domain sequence SEQ ID NO: 95 and a light chainvariable domain sequence selected from the group consisting of SEQ IDNO: 98 and SEQ ID NO:99.

In some embodiments, the humanized antibody comprises a heavy chainvariable domain sequence selected from the group consisting of SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,and SEQ ID NO: 107 (FIG. 26A; h2107). In some embodiments, the humanizedantibody comprises a light chain variable domain sequence selected fromthe group consisting of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO:112 (FIG. 26B; h2107). In another embodiment, the humanized antibodycomprises a light chain variable domain sequence SEQ ID NO: 111 and aheavy chain variable domain sequence selected from the group consistingof SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105.In one other embodiment, the humanized antibody comprises a heavy chainvariable domain sequence SEQ ID NO: 102 and a light chain variabledomain sequence selected from the group consisting of SEQ ID NO: 110 andSEQ ID NO: 112. In one embodiment, the humanized antibody comprises aheavy chain variable domain sequence SEQ ID NO: 106 and a light chainvariable domain sequence selected from the group consisting of SEQ IDNO: 110, SEQ ID NO: 111, and SEQ ID NO: 112. In one embodiment, thehumanized antibody comprises a heavy chain variable domain sequence SEQID NO: 107 and a light chain variable domain sequence selected from thegroup consisting of SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112.

In some embodiments, the humanized antibody comprises a heavy chainvariable domain sequence selected from the group consisting of SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO:119 (FIG. 27A; h2120). In some embodiments, the humanized antibodycomprises a light chain variable domain sequence selected from the groupconsisting of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123 (FIG.27B; h2120). In another embodiment, the humanized antibody comprises alight chain variable domain sequence SEQ ID NO: 123 and a heavy chainvariable domain sequence selected from the group consisting of SEQ IDNO: 115, SEQ ID NO: 116, and SEQ ID NO: 117. In one other embodiment,the humanized antibody comprises a light chain variable domain sequenceSEQ ID NO: 123 and a heavy chain variable domain sequence selected fromthe group consisting of SEQ ID NO: 108 and SEQ ID NO: 109. In oneembodiment, the humanized antibody comprises a heavy chain variabledomain sequence SEQ ID NO: 115 and a light chain variable domainsequence selected from the group consisting of SEQ ID NO: 121 and SEQ IDNO: 122.

In one embodiment, the invention provides an isolated humanized h1749anti-MCAM antibody, or antigen binding fragment thereof, comprising twoheavy chains (VH1 and VH2) and two light chains (VL1 and VL2). The VH1heavy chain comprises SEQ ID NO: 94, three heavy chain hypervariableregions (HVR-HC) comprising SEQ ID NOs: 66, 67, and 68, respectively,and four heavy chain frame work regions (FR-HC) comprising SEQ ID NOs:155, 125, 126, and 127, respectively. The VH2 heavy chain of h1749comprises SEQ ID NO: 95, three heavy chain hypervariable regions(HVR-HC) comprising SEQ ID NOs: 66, 67, and 68, respectively, and fourheavy chain frame work regions (FR-HC) comprising SEQ ID NOs: 155, 128,126, and 127, respectively. The VL1 light chain of h1749 comprises SEQID NO: 98, three light chain hypervariable regions (HVR-LC) comprisingSEQ ID NOs: 61, 62, and 63, respectively, and four light chain framework regions (FR-LC) comprising SEQ ID NOs: 128, 129, 130, and 131,respectively. The VL2 light chain of h1749 comprises SEQ ID NO: 99,three light chain hypervariable regions (HVR-LC) comprising SEQ ID NOs:61, 62, and 63, respectively, and four light chain frame work regions(FR-LC) comprising SEQ ID NOs: 128, 129, 132, and 131, respectively.

In another embodiment, the invention provides an isolated humanizedh2107 anti-MCAM antibody, or antigen binding fragment thereof,comprising six heavy chains (VH1, VH2, VH3, VH4, VH5, and VH6) and threelight chains (VL1, VL2, and VL3). The VH1 heavy chain comprises SEQ IDNO: 102, three heavy chain hypervariable regions (HVR-HC) comprising SEQID NOs: 90 or 151, 91, and 92, respectively, and four heavy chain framework regions (FR-HC) comprising SEQ ID NOs: 133, 134, 135, and 136,respectively. The VH2 heavy chain of h2107 comprises SEQ ID NO: 103,three heavy chain hypervariable regions (HVR-HC) comprising SEQ ID NOs:90 or 151, 91, and 92, respectively, and four heavy chain frame workregions (FR-HC) comprising SEQ ID NOs: 133, 134, 137, and 136,respectively. The VH3 heavy chain of h2107 comprises SEQ ID NO: 104,three heavy chain hypervariable regions (HVR-HC) comprising SEQ ID NOs:90 or 151, 91, and 92, respectively, and four heavy chain frame workregions (FR-HC) comprising SEQ ID NOs: 133, 134, 138, and 136,respectively. The VH4 heavy chain of h2107 comprises SEQ ID NO: 105,three heavy chain hypervariable regions (HVR-HC) comprising SEQ ID NOs:139 or 152, 91, and 92, respectively, and four heavy chain frame workregions (FR-HC) comprising SEQ ID NOs: 133, 134, 135, and 136,respectively. The VH5 heavy chain of h2107 comprises SEQ ID NO: 106,three heavy chain hypervariable regions (HVR-HC) comprising SEQ ID NOs:140, 91, 92, respectively, and four heavy chain frame work regions(FR-HC) comprising SEQ ID NOs: 133, 134, 135, and 136, respectively. TheVH6 heavy chain of h2107 comprises SEQ ID NO: 107, three heavy chainhypervariable regions (HVR-HC) comprising SEQ ID NOs: 141, 91, and 92,respectively, and four heavy chain frame work regions (FR-HC) comprisingSEQ ID NOs: 133, 134, 135, and 136, respectively.

The VL1 light chain of h2107 comprises SEQ ID NO: 110, three light chainhypervariable regions (HVR-LC) comprising SEQ ID NOs: 85, 86, and 87,respectively, and four light chain frame work regions (FR-LC) comprisingSEQ ID NOs: 142, 143, 144, and 145, respectively. The VL2 light chain ofh2107 comprises SEQ ID NO: 111, three light chain hypervariable regions(HVR-LC) comprising SEQ ID NOs: 85, 86, and 87, respectively, and fourlight chain frame work regions (FR-LC) comprising SEQ ID NOs: 142, 146,144, and 145, respectively. The VL3 light chain of h2107 comprises SEQID NO: 112, three light chain hypervariable regions (HVR-LC), comprisingSEQ ID NOs: 85, 86, and 87, respectively, and four light chain framework regions (FR-LC) comprising SEQ ID NOs: 147, 143, 144, and 145,respectively.

In another embodiment, the invention provides an isolated humanizedh2120 anti-MCAM antibody, or antigen binding fragment thereof,comprising five heavy chains (VH1, VH2, VH3, VH4, and VH5) and threelight chains (VL1, VL2, and VL3). The VH1 heavy chain comprises SEQ IDNO: 115, three heavy chain hypervariable regions (HVR-HC) comprising SEQID NOs: 78, 79, and 153, respectively, and four heavy chain frame workregions (FR-HC) comprising SEQ ID NOs: 133, 134, 135, and 136,respectively. The VH2 heavy chain of h2120 comprises SEQ ID NO: 116,three heavy chain hypervariable regions (HVR-HC) comprising SEQ ID NOs:78, 79, and 153, respectively, and four heavy chain frame work regions(FR-HC) comprising SEQ ID NOs: 133, 134, 138, and 136, respectively. TheVH3 heavy chain of h2120 comprises SEQ ID NO: 117, three heavy chainhypervariable regions (HVR-HC) comprising SEQ ID NOs: 139, 79, and 153,respectively, and four heavy chain frame work regions (FR-HC) comprisingSEQ ID NOs: 133, 134, 135, and 136, respectively. The VH4 heavy chain ofh2120 comprises SEQ ID NO: 118, three heavy chain hypervariable regions(HVR-HC) comprising SEQ ID NOs: 140, 79, and 153, respectively, and fourheavy chain frame work regions (FR-HC) comprising SEQ ID NOs: 133, 134,135, and 136, respectively. The VH5 heavy chain of h2120 comprises SEQID NO: 119, three heavy chain hypervariable regions (HVR-HC) comprisingSEQ ID NOs: 141, 79, and 153, respectively, and four heavy chain framework regions (FR-HC) comprising SEQ ID NOs: 133, 134, 135, and 136,respectively.

The VL1 light chain of h2120 comprises SEQ ID NO: 121, three light chainhypervariable regions (HVR-LC) comprising SEQ ID NOs: 73, 74, and 75,respectively, and four light chain frame work regions (FR-LC) comprisingSEQ ID NOs: 142, 148, 149, and 150, respectively. The VL2 light chain ofh2120 comprises SEQ ID NO: 122, three light chain hypervariable regions(HVR-LC) comprising SEQ ID NOs: 73, 74, and 75, respectively, and fourlight chain frame work regions (FR-LC) comprising SEQ ID NOs: 142, 148,154, and 150, respectively. The VL3 light chain of h2120 comprises SEQID NO: 123, three light chain hypervariable regions (HVR-LC) comprisingSEQ ID NOs: 73, 74, and 75, respectively, and four light chain framework regions (FR-LC) comprising SEQ ID NOs: 147, 148, 149, and 150,respectively.

In some embodiments, the invention provides an antibody that binds toMCAM, wherein the antibody comprises a heavy chain variable domainhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to an amino acid sequence selected from SEQ ID NO:94, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104; SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119. In someembodiments, the anti-MCAM antibody comprises a light chain variabledomain having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO:123. In some embodiment, the antibody comprises a heavy chain variabledomain having at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence selected from SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104; SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, and SEQ ID NO: 119, and theantibody further comprises a light chain variable domain having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

In some embodiments, the invention provides an antibody that is avariant of any of the above antibodies having one or more amino acidsubstitutions, deletions, insertions or modifications, and which retainsa biological function of the antibody. In some embodiments, theinvention provides an antibody that binds to MCAM expressed on the cellsurface and inhibits the binding of MCAM to laminin 411. In someembodiments, the anti-MCAM antibody binds to MCAM expressed on the cellsurface and inhibits disease progression. In some embodiments, theprogression of an autoimmune disease is inhibited. In one embodiment,the progression of multiple sclerosis is inhibited. In some embodiments,the invention provides an antibody that is a variant of any one of theabove antibodies having improvements in one or more of a property suchas binding affinity, specificity, thermostability, expression level,effector function, glycosylation, reduced immunogenicity, or solubilityas compared to the unmodified antibody.

In other embodiments, the anti-MCAM antibody binds to MCAM expressed onthe cell surface and inhibits progression of a metastatic cancer. In oneembodiment, the metastatic cancer is selected from the group consistingof prostate cancer, lung cancer, and pancreatic cancer.

The invention herein includes the production and use of MCAM antagonistantibodies. Exemplary methods for generating antibodies are described inmore detail herein. MCAM antibodies can include, but are not limited to,polyclonal, monoclonal, multispecific, human, humanized, primatized, orchimeric antibodies, single chain antibodies (e.g., scFv), Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the present embodiments), andepitope-binding fragments of any of the above. Human antigen-bindingantibody fragments include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), and fragments comprising either a VL or VHdomain. Antigen-binding antibody fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,and CH3 domains. Also included are antigen-binding fragments that cancomprise any combination of variable region(s) with a hinge region, CH1,CH2, and CH3 domains. The antibodies may be from any animal originincluding birds and mammals. Typically, the antibodies are from human orother primates, murine (e.g., mouse and rat), donkey, sheep, monkey,rabbit, goat, guinea pig, pig, camel, horse, or chicken (or otheravian). As used herein, “human” antibodies include antibodies having theamino acid sequence of a human immunoglobulin and include antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulins and that do not express endogenousimmunoglobulins, as described, for example in, U.S. Pat. No. 5,939,598.

In another embodiment, the MCAM antibody can be a monoclonal antibody.In yet a further embodiment, the antibody may be chemically modified,e.g., by pegylation. Additionally, other antibodies can be identifiedusing techniques available in the art. For example, antibodies capableof specifically binding to MCAM can be produced using phage displaytechnology. Antibody fragments that selectively bind to MCAM can then beisolated. Exemplary methods for producing such antibodies via phagedisplay are disclosed, for example, in U.S. Pat. No. 6,225,447, forexample.

Monoclonal antibodies can also be produced using the conventionalhybridoma methods. These methods have been widely applied to producehybrid cell lines that secrete high levels of monoclonal antibodiesagainst many specific antigens, and can also be used to producemonoclonal antibodies capable of specifically binding to MCAM. Forexample, mice (e.g., Balb/c mice) can be immunized with an antigenicMCAM epitope by intraperitoneal injection. After sufficient time haspassed to allow for an immune response, the mice are sacrificed, and thespleen cells obtained and fused with myeloma cells, using techniqueswell known in the art. The resulting fused cells, hybridomas, are thengrown in a selective medium, and the surviving cells grown in suchmedium using limiting dilution conditions. After cloning and recloning,hybridomas can be isolated for secreting antibodies (for example, of theIgG or IgM class or IgG1 subclass) that selectively bind to MCAM. Toproduce agents specific for human use, the isolated monoclonal can thenbe used to produce chimeric and humanized antibodies.

MCAM antagonist antibodies are selected using an antigen derived from amammalian species. Preferably the antigen is human MCAM or a laminin α4chain, e.g., α4 chain of laminin 411. However, polypeptides from otherspecies such as murine MCAM or laminin α4 chain can also be used as thetarget antigen. The antigens from various mammalian species may beisolated from natural sources. In other embodiments, the antigen isproduced recombinantly or made using other synthetic methods known inthe art. The antibody selected will normally have a sufficiently strongbinding affinity for the antigen. For example, the antibody may bindhuman MCAM or a laminin α4 chain, e.g., an α4 chain of laminin 411 witha K_(d) value of no more than about 5 nM, preferably no more than about2 nM, and more preferably no more than about 500 pM. Antibody affinitiesmay be determined by a surface plasmon resonance based assay (such asthe BIAcore assay as described in Examples); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample.

Also, the antibody may be subject to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody. Examples include the experimentalautoimmune encephalomyelitis (EAE) (as described in Example 7 below),and in vitro and in vivo assays described herein for identifying MCAMantagonists.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest.

In a preferred embodiment, the antagonist antibodies are selected usinga unique phage display approach. The approach involves generation ofsynthetic antibody phage libraries based on single framework template,design of sufficient diversities within variable domains, display ofpolypeptides having the diversified variable domains, selection ofcandidate antibodies with high affinity to target antigen, and isolationof the selected antibodies. Details of the phage display methods can befound, for example, in WO03/102157 published Dec. 11, 2003. The antibodygenerated from phage libraries can be further modified to generateantibody mutants with improved physical, chemical and or biologicalproperties over the parent antibody. Where the assay used is abiological activity assay, the antibody mutant preferably has abiological activity in the assay of choice which is at least about 10fold better, preferably at least about 20 fold better, more preferablyat least about 50 fold better, and sometimes at least about 100 fold or200 fold better, than the biological activity of the parent antibody inthat assay. For example, an anti-MCAM antibody mutant preferably has abinding affinity for MCAM which is at least about 10 fold stronger,preferably at least about 20 fold stronger, more preferably at leastabout 50 fold stronger, and sometimes at least about 100 fold or 200fold stronger, than the binding affinity of the parent anti-MCAMantibodies, such as clone 15 or 17 antibodies.

Chimeric and humanized antibodies can be produced from non-humanantibodies, and can have the same or similar binding affinity as theantibody from which they are produced. Exemplary techniques forproducing chimeric antibodies include splicing the genes from, e.g., amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activity.See, e.g., Morrison et al., 1984 Proc. Nat'l. Acad. Sci. USA 81: 6851;Neuberger et al., 1984 Nature 312: 604; and Takeda et al., 1985 Nature314: 452. For example, a nucleic acid encoding a variable (V) region ofa mouse monoclonal antibody can be joined to a nucleic acid encoding ahuman constant (C) region, e.g., IgG1 or IgG4. The resulting antibody isthus a species hybrid, generally with the antigen binding domain fromthe non-human antibody and the C or effector domain from a human orprimate antibody.

Humanized antibodies are antibodies with variable regions that areprimarily from a human antibody (i.e., the acceptor antibody), but whichhave complementarity determining regions substantially from a non-humanantibody (the donor antibody). See, e.g., Queen et al., Proc. Nat'l.Acad. Sci USA 86: 10029-10033 (1989); WO 90/07861, U.S. Pat. Nos.7,435,802, 6,054,297; 5,693,761; 5,585,089; 5,530,101; and 5,224,539.The constant region or regions of these antibodies are generally alsofrom a human antibody. The human variable domains are typically chosenfrom human antibodies having sequences displaying a high homology withthe desired non-human variable region binding domains. The heavy andlight chain variable residues can be derived from the same antibody, ora different human antibody. In addition, the sequences can be chosen asa consensus of several human antibodies, such as described in WO92/22653.

A “Primatized™ antibody” is a recombinant antibody containing primatevariable sequences or antigen binding portions, and human constantdomain sequences. See e.g., Newman, Bio/Technology, 1992, 10: 1455-60.Primatization of antibodies results in the generation of antibodies thatcontain primate (e.g., monkey) variable domains and human constantsequences. See, e.g., U.S. Pat. No. 6,113,898. This technique modifiesantibodies such that they are not rejected upon administration in humansbecause they are antigenic. This technique relies on immunization ofcynomolgus monkeys with human antigens or receptors. This technique wasdeveloped to create high affinity monoclonal antibodies directed tohuman cell surface antigens.

In another aspect, specific amino acids within the human variable regioncan be selected for substitution based on the predicted conformation andantigen binding properties. This can be determined using techniques suchas computer modeling, prediction of the behavior and binding propertiesof amino acids at certain locations within the variable region, andobservation of effects of substitution. For example, when an amino aciddiffers between a non-human variable region and a human variable region,the human variable region can be altered to reflect the amino acidcomposition of the non-human variable region. In a specific embodiment,the antibodies used in the chronic dosage regime can be humanizedantibodies as disclosed in U.S. Pat. No. 5,840,299. In anotherembodiment, transgenic mice containing human antibody genes can beimmunized with an antigenic MCAM structure and hybridoma technology canbe used to generate human antibodies that selectively bind to MCAM.

Chimeric, human and/or humanized antibodies can be produced by usingrecombinant expression, e.g., expression in human hybridomas (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985)), in myeloma cells, or in Chinese hamster ovary (CHO) cells.Alternatively, antibody coding sequences can be incorporated intotransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal. See, e.g.,U.S. Pat. No. 6,197,946. Exemplary suitable transgenes include, but arenot limited to, transgenes having a promoter and/or enhancer from amammary gland specific gene, for example casein or β-lactoglobulin.

6.3 Antibody Variants

In addition to the MCAM antagonist antibodies described herein, it iscontemplated that variants of such antibodies can be prepared. Anti-MCAMantagonist antibody variants can be prepared by introducing appropriatenucleotide changes into the encoding DNA, and/or by synthesis of thedesired antibody. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of the anti-MCAMantibody, such as changing the number or position of glycosylationsites.

Variations in the MCAM antagonist antibodies described herein, can bemade, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the antibody that results in achange in the amino acid sequence as compared with the native sequenceantibody. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe MCAM antagonist antibody. Guidance in determining which amino acidresidue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe MCAM antagonist antibody with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Covalent modifications of anti-MCAM antibodies are included within thescope of this invention. Covalent modifications include reactingtargeted amino acid residues of an anti-MCAM antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the anti-MCAM antibody. Othermodifications include deamidation of glutaminyl and asparaginyl residuesto the corresponding glutamyl and aspartyl residues, respectively,hydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation ofany C-terminal carboxyl group.

In one aspect, the MCAM antagonist antibodies of the present inventioncomprise one or more deamidation mutations in the amino acid sequence.The deamidation of amino acid residues is a common structuralmodification in recombinant polypeptides, which can lead to theformation of iso-aspartic acid resulting in decreased stability.Deamidation may be associated with glycine (G)-asparginine (N)sequences, including G-N and N-G sequences. In one embodiment, theantibody comprises a deamidation mutation. In another embodiment, thedeamidation mutation is the substitution of an N amino acid residue or aG amino acid residue. In some embodiments, the substitution is selectedfrom the group consisting of N→S, N→A, and G→Q. In one embodiment, thedeamidation mutation is located at Kabat residue N32 or G33.

Other types of covalent modification of the anti-MCAM antibody includedwithin the scope of this invention include altering the nativeglycosylation pattern of the antibody or polypeptide (Beck et al., Curr.Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug Discov. Today 15:773-780, 2010), and linking the antibody to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

In one aspect, the MCAM antagonist antibodies of the present inventioncomprise one or more glycosylation mutations in the amino acid sequence.The mutation may remove or restore/introduce a glycosylation site.Generally, the glycosylation mutation is associated with an asparginine(N) residue. In one embodiment, the antibody comprises a glycosylationmutation. In another embodiment, the glycosylation mutation is theintroduction of an N amino acid residue. In some embodiments, theintroduction is the replacement of an aspartic acid (D) residue with anN residue (D→N). In one embodiment, the mutation is located at Kabatresidue 72.

MCAM antagonist antibody fragments are provided herein. Such fragmentsmay be truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native antibody.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the MCAM antagonist antibody.

MCAM antagonist antibody fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating antibody orpolypeptide fragments by enzymatic digestion, e.g., by treating theprotein with an enzyme known to cleave proteins at sites defined byparticular amino acid residues, or by digesting the DNA with suitablerestriction enzymes and isolating the desired fragment. Yet anothersuitable technique involves isolating and amplifying a DNA fragmentencoding a desired antibody or polypeptide fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, anti-MCAM antagonist antibody fragments share at least onebiological and/or immunological activity with a native MCAM antagonistantibody disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 1 below under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, as further described below in reference to aminoacid classes, are introduced and the products screened.

Substantial modifications in function or immunological identity of theMCAM antagonist antibody are accomplished by selecting substitutionsthat differ significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

TABLE 1 Preferred Original Residue Exemplary Substitutions SubstitutionsArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucineleu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln;asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leuPro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucineleu

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the MCAM antagonist antibody variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the MCAM antagonist antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theMCAM antagonist antibody to improve its stability (particularly wherethe antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and human MCAM or laminin 411polypeptide. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Preferred affinity matured antibodies have an affinity which is fivetimes, more preferably 10 times, even more preferably 20 or 30 timesgreater than the starting antibody (generally murine, humanized orhuman) from which the matured antibody is prepared.

Nucleic acid molecules encoding amino acid sequence variants of the MCAMantagonist antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the MCAM antagonistantibody.

Also included in the invention are antibodies that bind to the sameepitope as the antibodies described herein. For example, antibodies ofthe invention specifically bind to an epitope that includes one or moreamino acid residues on human MCAM (Accession No. AAA20922.1/CAA48332).In some embodiments, antibodies of the invention specifically bind MCAM,wherein the antibody binds to an epitope on human MCAM (e.g., AccessionNo. AAA20922.1/CAA48332).

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., fully human monoclonal antibody) has the same specificity as amonoclonal antibody of the invention by ascertaining whether the formerprevents the latter from binding to MCAM. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention, then the two monoclonal antibodies bind to the same, or aclosely related, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with MCAM (e.g.,an MCAM-Fc molecule exemplified in the Examples) and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind MCAM. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

In a further embodiment, the invention comprises a binding agent thatbinds to essentially the same epitope as any of the antibodies disclosedherein. In one embodiment, the binding agent is capable of binding toMCAM protein on the surface of cells. In some embodiments, the bindingagent inhibits the interaction of MCAM (e.g., cell surface MCAM) withits ligand, a protein comprising a laminin alpha-4 chain. In someembodiments, the binding agent is an antibody or a functional fragmentthereof.

In one embodiment, the invention provides a binding agent capable ofbinding to MCAM, wherein any one of the antibodies disclosed abovedisplaces the binding agent in a competitive binding assay. In someembodiments, the binding agent is an antibody, or a functional fragmentthereof. In another embodiment, the invention provides a binding agentcapable of binding to MCAM, wherein the binding agent displaces any oneof the antibodies disclosed above in a competitive binding assay. Insome embodiments, the binding agent is an antibody, or a functionalfragment thereof.

In other embodiments, the binding agent is an alternative binding agent.These alternative binding agents may include, for example, any of theengineered protein scaffolds known in the art. Such scaffolds include,for example, anticalins, which are based upon the lipocalin scaffold, aprotein structure characterized by a rigid beta-barrel that supportsfour hypervariable loops which form the ligand binding site. Novelbinding specificities are engineered by targeted random mutagenesis inthe loop regions, in combination with functional display and guidedselection (Skerra (2008) FEBS J. 275: 2677-2683). Other suitablescaffolds may include, for example, adnectins, or monobodies, based onthe tenth extracellular domain of human fibronectin III (Koide and Koide(2007) Methods Mol. Biol. 352: 95-109); affibodies, based on the Zdomain of staphylococcal protein A (Nygren et al. (2008) FEBS J. 275:2668-2676)); DARPins, based on ankyrin repeat proteins (Stumpp et al.(2008) Drug. Discov. Today 13: 695-701); fynomers, based on the SH3domain of the human Fyn protein kinase (Grabulovski et al. (2007) J.Biol. Chem. 282: 3196-3204); affitins, based on Sac7d from Sulfolobusacidolarius (Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058-1068);affilins, based on human y-B-crystallin (Ebersbach et al. (2007) J. Mol.Biol. 372: 172-185); avimers, based on the A domains of membranereceptor proteins (Silverman et al. (2005) Biotechnol. 23: 1556-1561);cysteine-rich knottin peptides (Kolmar (2008) FEBS J. 275: 2684-2690);and engineered Kunitz-type inhibitors (Nixon and Wood (2006) Curr. Opin.Drug. Discov. Dev. 9: 261-268). For review, see Gebauer and Skerra(2009) Curr. Opin. Chem. Biol. 13: 245-255. In other embodiments, thebinding agent is an anticalin, an adnectin, an affibody, a DARPin, afynomer, an affitin, an affilin, an avimer, a cysteine-rich knottinpeptide, or an engineered Kunitz-type inhibitor.

7. Methods of Use

The present invention provides MCAM antagonists as therapeutic agentsfor neuroinflammatory conditions, cancer, and autoimmune diseases. Forthe prevention, treatment or reduction in the severity of a givendisease or condition, the appropriate dosage of a compound of theinvention will depend on the type of disease or condition to be treated,as defined above, the severity and course of the disease or condition,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the compound, and the discretion of the attending physician. Thecompound is suitably administered to the patient at one time or over aseries of treatments. Preferably, it is desirable to determine thedose-response curve and the pharmaceutical composition of the inventionfirst in vitro, and then in useful animal models prior to testing inhumans.

In one aspect, the present invention provides a method for inhibiting orblocking the interaction of MCAM expressed on T cells and laminin α4chain, e.g., an α4 chain of laminin 411, comprising treating the T cellswith an MCAM antagonist (as described herein), thereby inhibiting theinteraction of MCAM with laminin α4 chain. In one embodiment, thelaminin α4 chain is expressed on the surface of a cell, e.g., anendothelial cell. In a preferred embodiment, the MCAM antagonist is ananti-MCAM antibody. In another embodiment, the T cells are TH17 cells.In one other embodiment, the treatment with an MCAM antagonist isperformed in vivo. In yet another embodiment, the treatment is performedin a mammalian subject. In one embodiment, the mammalian subject is ahuman.

In another aspect, the present invention provides a method forinhibiting or preventing extravasation of MCAM-expressing T cells intothe central nervous system (CNS) comprising treating the T cells with anMCAM antagonist (as described herein), thereby inhibiting or preventingthe extravasation of MCAM-expressing T cells into the CNS. In oneembodiment, the MCAM antagonist blocks the interaction of MCAM withlaminin α4 chain, e.g., an α4 chain of laminin 411. In a preferredembodiment, the MCAM antagonist is an anti-MCAM antibody. In one otherembodiment, the laminin α4 chain is expressed on the surface of a cell,e.g., an endothelial cell. In another embodiment, the T cells are TH17cells. In one other embodiment, the treatment with an MCAM antagonist isperformed in vivo. In yet another embodiment, the treatment is performedin a mammalian subject. In one embodiment, the mammalian subject is ahuman.

In one other aspect, the present invention provides methods of treatmentfor a neuroinflammatory condition, a cancerous condition, or anautoimmune disease. In one embodiment, the method comprisesadministering to a mammalian subject in need a therapeutically effectiveamount of an MCAM antagonist. In another aspect, the invention providesa method for the delaying or slowing down of the progression of aneuroinflammatory condition, a cancerous condition, or an autoimmunedisease. In one embodiment, the method comprises administering tosubject diagnosed with the condition or disease, an effective amount ofan MCAM antagonist. In another aspect, the invention provides a methodfor preventing indicia of a neuroinflammatory condition, cancerouscondition, or an autoimmune disease. In one embodiment, the methodcomprises administering an effective amount of an MCAM antagonist to asubject at risk of the condition or disease, wherein the MCAM antagonistis effective against the development of indicia of the condition ordisease. In one additional aspect, the present invention providesmethods of treatment for a metastatic cancer.

In one embodiment, the present invention provides an MCAM antagonist foruse as a medicament for, or for the treatment of a disease, condition ordisorder described herein. In another embodiment, the present inventionprovides the use of an MCAM antagonist for the manufacture of amedicament for treating a disease, condition or disorder describedherein. In one other embodiment, the present invention provides the useof an MCAM antagonist described herein, in the manufacture of amedicament for the treatment of a central nervous system (CNS)inflammatory disorder characterized by infiltration of MCAM-expressingcells into the CNS.

7.1 Neuroinflammatory Conditions

In one aspect, the MCAM antagonists provide a preventative orprophylactic effect against the development of, or the progression of,clinical and/or histological and/or biochemical and/or pathologicalindicia (including both symptoms and signs) of neuroinflammatoryconditions in a subject. In one embodiment, the neuroinflammatorycondition is characterized by CNS inflammation and/or cell/tissuedamage. In one embodiment, the indicia include increased glialactivation, increased pro-inflammatory cytokine/chemokine levels (e.g.,TNFα, INFγ, IL-1β), increased blood-brain-barrier permeability, and/orincreased immune cell (e.g., leukocyte) recruitment/invasion to the CNS.In another embodiment, the neuroinflammation is progressive or chronicneuroinflammation associated with chronic activation of cells of theimmune system (i.e., autoimmune-associated neuroinflammation). Chronicneuroinflammation conditions include, without limitation, relapsingmultiple sclerosis (MS), chronic progressive MS, inactive MS, andParkinson's disease (PD). In another embodiment, the subject is at riskfor a neuroinflammatory condition. In general, a subject at risk willpreviously have had a neuroinflammatory condition as described herein,or will have a genetic predisposition for neuroinflammatory condition.

The efficacy of the treatment of neuroinflammatory conditions can bemeasured by various assessments commonly used in evaluatingneuroinflammatory condition. For example, CNS health can be evaluated bytesting for MS symptoms including, but not limited to, impaired vision(e.g., blurred or double vision, red-green color distortion, orblindness); muscle weakness in the extremities; impaired coordinationand balance; partial or complete paralysis, paresthesias, transitoryabnormal sensory feelings (e.g., numbness, prickling, or “pins andneedles” sensations); pain; speech impediments; tremors; dizziness;hearing loss; cognitive impairments (e.g., difficulties withconcentration, attention, memory, and poor judgment); and depression. MStesting may also include a lumbar puncture (spinal tap) forcerebrospinal fluid (CSF) tests (e.g., CSF oligoclonal bandingsuggesting inflammation of the CNS); a magnetic resonance imaging (MRI)scan of the head or spine; and a nerve function test (e.g., evokedpotential test).

CNS health may also be evaluated by testing for PD symptoms including,but not limited to, tremor (e.g., trembling in hands, arms, legs, jaw,and face); rigidity or stiffness of the limbs and trunk; bradykinesia orslowness of movement; postural instability or impaired balance andcoordination; depression and other emotional changes; difficulty inswallowing, chewing, and speaking; urinary problems or constipation;skin problems; sleep disruptions; and brain scans or other tests to ruleout other diseases.

7.2 Autoimmune Diseases

For autoimmune diseases, the term “treatment” refers to both therapeutictreatment and prophylactic or preventative measures for an autoimmunedisease, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. Those in need of treatmentinclude those already with an autoimmune disease as well as those proneto have an autoimmune disease or those in whom the autoimmune disease isto be prevented.

In one aspect, the MCAM antagonists provide a preventative orprophylactic effect against the development of, or the progression of,clinical and/or histological and/or biochemical and/or pathologicalindicia (including both symptoms and signs) of autoimmune disease in asubject. In another embodiment, the subject is at risk for autoimmunedisease or an autoimmune disease flare-up. In general, a subject at riskwill previously have had autoimmune disease and/or one or moreautoimmune disease flare-ups, or will have a genetic predisposition foran autoimmune disease.

7.3 Metastatic Cancers

For metastatic cancer, the term “treatment” refers to both therapeutictreatment and prophylactic or preventative measures for a metastaticcancer, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. Those in need of treatmentinclude those already with a metastatic cancer as well as those prone tohave a metastatic cancer or those in whom the metastatic cancer is to beprevented.

In one aspect, the MCAM antagonists provide a preventative orprophylactic effect against the development of, or the progression of,clinical and/or histological and/or biochemical and/or pathologicalindicia (including both symptoms and signs) of a metastatic cancer in asubject. In one embodiment, the metastatic cancer is selected from thegroup consisting of prostate cancer, lung cancer, and pancreas cancer.

7.4 Combination Therapy

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is one or more of the following disease-modifying agents:teriflunomide, interferon beta-1a, interferon beta-1b, glatirameracetate, fingolimod, and mitoxantrone. In another embodiment, anadditional therapeutic agent is an agent which treats an acuteexacerbation of a disease. In one embodiment, the additional therapeuticagent for acute exacerbation is one or more corticosteroids. In oneother embodiment, the one or more corticosteroids are selected from thegroup consisting of prednisone, methylprednisolone, and dexamethasone.In another embodiment, the disease is multiple sclerosis and the acuteexacerbation is a relapse or attack (e.g., inflammation of the CNS).

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent.

8. Pharmaceutical Compositions

MCAM antagonist antibodies specifically binding MCAM or a laminin α4chain, e.g., an α4 chain of laminin 411, as well as other MCAMantagonist molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of variousdisorders, in particular neuroinflammatory diseases or diseasesbenefiting from the inhibition of the infiltration of MCAM-expressingcells into the CNS, in the form of pharmaceutical compositions.

In one aspect, the present invention concerns pharmaceuticalcompositions comprising an antibody, or antigen binding fragmentthereof, as described herein. In one embodiment, the pharmaceuticalcomposition comprises

-   -   (i) an isolated anti-MCAM antibody, or antigen binding fragment        thereof, that binds to an immunoglobulin domain of MCAM        comprising the amino acid sequence shown as SEQ ID NO:22;    -   (ii) an isolated anti-MCAM antibody, or antigen binding fragment        thereof, that binds to an immunoglobulin domain of MCAM        comprising the amino acid sequence shown as SEQ ID NO:23; or    -   (iii) an isolated anti-MCAM antibody, or antigen binding        fragment thereof, that binds to a domain of MCAM comprising the        amino acid sequences shown as SEQ ID NOS: 22 and 23.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence KASKNIDTYLA        (SEQ ID NO:3);    -   (ii) an HVR-L2 comprising the amino acid sequence SGSTL (SEQ ID        NO:4);    -   (iii) an HVR-L3 comprising the amino acid sequence QQHNEYPLT        (SEQ ID NO:5);    -   (iv) an HVR-H1 comprising the amino acid sequence GFTFSNYYMA        (SEQ ID NO:8)    -   (v) an HVR-H2 comprising the amino acid sequence        SISFEGNRNHYGDSVK (SEQ ID NO:9); and/or    -   (vi) an HVR-H3 comprising the amino acid sequence        HRGYSTNFYHDVLDAWGQG (SEQ ID NO:10).

In one other embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence        KSSQSLLYSGTQKNYLA (SEQ ID NO:14);    -   (ii) an HVR-L2 comprising the amino acid sequence WASTRQS (SEQ        ID NO:15);    -   (iii) an HVR-L3 comprising the amino acid sequence QQYYDTLTDT        (SEQ ID NO:16);    -   (iv) an HVR-H1 comprising the amino acid sequence GFKFSNYYMS        (SEQ ID NO:19);    -   (v) an HVR-H2 comprising the amino acid sequence        SISDGGGDTFCRDLVKG (SEQ ID NO:20); and/or    -   (vi) an HVR-H3 comprising the amino acid sequence        RGAAMGGVMDAWGQG (SEQ ID NO:21).

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:31;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:32;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:33;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:36;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:37; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:38.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:41;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:42;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:43;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:46;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:47; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:48.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:51;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:52;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:53;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:56;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:57; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:58.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:61;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:62;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:63;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:66;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:67; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:68.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:73;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:74;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:75;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:78;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:79; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:80.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising the following hypervariable regions (HVRs):

-   -   (i) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:85;    -   (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:86;    -   (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:87;    -   (iv) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:90;    -   (v) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:91; and/or    -   (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:92.

In another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof,comprising

-   -   (a) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:2 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:7;    -   (b) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:13 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:18;    -   (c) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:30 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:35;    -   (d) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:40 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:45;    -   (e) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:50 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:55;    -   (f) a light chain variable domain comprising the amino acid        sequence shown as SEQ ID NO:60 and a heavy chain variable domain        comprising the amino acid sequence shown as SEQ ID NO:65;    -   (g) a light chain variable domain comprising the amino acid        sequence shown as any one of SEQ ID NOS:70, 71, or 72 and a        heavy chain variable domain comprising the amino acid sequence        shown as SEQ ID NO:77; or    -   (h) a light chain variable domain comprising the amino acid        sequence shown as any one of SEQ ID NOS:83 or 84 and a heavy        chain variable domain comprising the amino acid sequence shown        as SEQ ID NO:89.

In yet another embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof, whichbinds to substantially the same epitope as an antibody described herein.In one other embodiment, the pharmaceutical composition comprises anisolated anti-MCAM antibody, or antigen binding fragment thereof, thatcompetes for binding to human MCAM with an antibody described herein. Inadditional embodiments, the present invention provides the use of ananti-MCAM antibody, or antigen binding fragment thereof, as describedherein, in the manufacture of a medicament for the treatment of acentral nervous system (CNS) inflammatory disorder characterized byinfiltration of MCAM-expressing cells into the CNS.

The compounds of the invention for prevention or treatment of aneuroinflammatory condition or autoimmune disease are typicallyadministered by intravenous injection. Other methods administration byalso be used, which includes but is not limited to, topical, parenteral,subcutaneous, intraperitoneal, intrapulmonary, intranasal, ocular,intraocular, intravitreal, intralesional, intracerobrospinal,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation administration. Parenteral infusions include intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneousadministration. In addition, the compounds described herein areadministered to a human subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time.

The present invention provides dosages for the MCAM antagonist-basedtherapeutics. For example, depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide isan initial candidate dosage for administration to the patient, whether,for example, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

The MCAM antagonist (including MCAM antagonist antibody) compositionsherein will be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the antagonist to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat a given disease or condition.

In some embodiments, the composition is used to prevent the occurrenceor reoccurrence of the disease or condition disease in the subject. Inone embodiment, the present invention can be used for increasing theduration of survival of a human patient susceptible to or diagnosed withthe disease or condition disease. Duration of survival is defined as thetime from first administration of the drug to death.

Therapeutic formulations are prepared using standard methods known inthe art by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (see, e.g., Alfonso R Gennaro (ed), Remington: The Scienceand Practice of Pharmacy, formerly Remington's Pharmaceutical Sciences20th ed., Lippincott, Williams & Wilkins, 2003, incorporated herein byreference in its entirety). Acceptable carriers, include saline, orbuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagines, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations.

Optionally, the formulations of the invention can contain apharmaceutically acceptable preservative. In some embodiments thepreservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben arepreferred preservatives. Optionally, the formulations of the inventioncan include a pharmaceutically acceptable surfactant at a concentrationof 0.005 to 0.02%.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

9. Articles of Manufacture and Kits

The instant invention further includes kits comprising the MCAMantagonists of the invention and related materials, such as instructionsfor use. The instructions for use may contain, for example, instructionsfor administration of the MCAM antagonists and optionally one or moreadditional agents. The invention also provides kits for the treatment ofa central nervous system (CNS) inflammatory disorder characterized byinfiltration of MCAM-expressing cells into the CNS. The disordersinclude, without limitation, neuroinflammatory conditions, such as, forexample, multiple sclerosis and Parkinson's disease, and autoimmunedisease. The kits of the invention comprise one or more containers of atleast one MCAM antagonist, preferably an antibody, in combination with aset of instructions, generally written instructions, relating to the useand dosage of the MCAM antagonist for the treatment of the disorder. Theinstructions included with the kit generally include information as todosage, dosing schedule, and route of administration for the treatmentof the target disorder, such as a neuroinflammatory condition or anautoimmune disease. The containers of MCAM antagonist(s) may be unitdoses, bulk packages (e.g., multi-dose packages), or sub-unit doses.

In one aspect, the present invention provides a kit comprising an MCAMantagonist as described herein and instructions for use in the treatmentof a central nervous system (CNS) inflammatory disorder characterized byinfiltration of MCAM-expressing cells into the CNS. In one embodiment,the present invention provides a kit for the treatment of a centralnervous system (CNS) inflammatory disorder characterized by infiltrationof MCAM-expressing cells into the CNS, said kit comprising: (a) acontainer comprising an MCAM antagonist antibody; and (b) a label orinstructions for administering said antibody to treat said CNSinflammatory disorder. Preferably, the CNS inflammatory disorder is aneuroinflammatory condition or an autoimmune disease. In one embodiment,the CNS inflammatory disorder is multiple sclerosis or Parkinson'sdisease.

Also provided is an article of manufacture for therapeutic use,comprising a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). At least one active agent in the composition is an MCAMantagonist of the invention. The label or package insert indicates thatthe composition is used for treating the particular condition. The labelor package insert will further comprise instructions for administeringthe antibody composition to the patient. Articles of manufacture andkits comprising combinatorial therapies described herein are alsocontemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

All patents, patent applications, and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

EXAMPLES

The following examples are not to be interpreted as limiting, but areexemplary means of using the methods disclosed.

Materials and Methods

Animals and Manipulation of Cells

SJL mice (Jackson), 8-16 week old, were immunized with PLP 139-151peptide emulsified in CFA. The commercial kit, EK-0122 (HookeLaboratories) was used for this immunization experiment. For someexperiments, spleens were removed 11 days later and processed into asingle cell suspension. For some experiments, splenocytes were processedfor in vitro analysis as described below. For EAE studies, mice wereinjected on days 5, 9, 13, and 17 after PLP immunization with eitherPBS, isotype control antibody (BioXcell), or anti-MCAM clone 17.Progression of the disease was monitored daily and scored in a blindedfashion by standard techniques. Mice were sacrificed 35 days after PLPimmunization, and brains and spinal cords were analyzed for infiltrationof immune cells.

For analysis of MCAM-Fc binding to EAE tissues, 8-16 week old C57BL6mice were immunized with myelin oligodendrocyte glycoprotein (MOG) 35-55emulsified in CFA. The commercial kit, EK-0111 (Hooke laboratories) wasused for this immunization experiment. The immunized animals weresacrificed at the peak of disease. Brains and spinal cords were snapfrozen in OCT (optimal cutting temperature media) and analyzed byfluorescent microscopy as described below.

Flow Cytometry/Marker Staining and Detection/FACS Protocols

Buffy coats were obtained from healthy human donors (Stanford BloodCenter, Palo Alto, Calif.) and CD4 T cells were negatively enrichedusing RosetteSep (Stem Cell Technologies). Where indicated, CD4+/CD45RO+memory T cells were further negatively purified using magnetic beads(Miltenyi Biotec). T cells were plated (2×10⁵ cells/well) in anti-CD3 (5μg/ml, BD Pharmingen) coated 96 well U bottom plates in RPMI containing10% heat-inactivated FCS (HyClone Laboratories), penicillin,streptomycin, L-glutamine, anti-IFNγ (5 μg/ml; R&D Systems), anti-IL4(0.5 μg/ml, R&D Systems), and anti-CD28 (2 μg/ml; BD Pharmingen) forfive days. Where indicated, TGFβ (2 ng/ml, unless otherwise indicated),IL12, IL1β, and/or IL-23 (all at 20 ng/ml) were added. All cytokineswere obtained from R&D Systems. Analysis of intracellular cytokinesoccurred following five hours in the presence of PMA (50 ng/ml) andIonomycin (500 ng/ml; both from Sigma-Aldrich) and GolgiStop (BDPharmingen). Surface staining with anti-MCAM (Pharmingen) was followedby fixation, permeabilization, and staining with anti-IL-17A(Ebioscience), IL-22 (R&D Systems), CCL20 (R&D Systems) and/or FOXP3using a FOXP3 staining kit (Biolegend). In some experiments,unmanipulated whole blood was stained for surface expression withanti-CCR7, anti-CCR6, anti-integrin alpha 4, anti-integrin beta 7, oranti-integrin beta 1 (all from BD Pharmingen).

Antibody Generation/Characterization

For the generation of antibodies capable of binding to murine MCAM,MCAM-Fc was generated by fusing the extracellular domain of murine MCAMto human IgG and produced in CHO cells using standard techniques. Lou/Mrats were immunized with 100 μg of MCAM-Fc protein in CFA (1:1 volume).Rats were boosted two times at two week intervals with MCAM-Fc proteinin incomplete Freund's adjuvant (IFA) (1:1 volume). Hybridomas weregenerated from immunized rats using standard protocols and clones wereselected by Clonepix. CHO cells were transfected with the full lengthmurine MCAM gene and selected for stable expression using neomycin andstandard techniques. Parental CHO cells (MCAM negative) werefluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE)using standard techniques and mixed at a 1:1 ratio with unlabeled MCAMtransfected CHO cells. Hybridoma supernatants were incubated with thismixture of cells for 30 minutes and binding of potential MCAM specificantibodies was detected with a fluorescently labeled anti-rat secondaryantibody (Jackson Immuno) by flow cytometry.

Supernatants from hybridomas that screened positive for MCAM specificantibodies were pre-incubated with fluorescently labeled mouse MCAM-Fcprotein (5 μg/mL) for 30 minutes before addition to the laminin α4expressing cell line WM2664 and neutralization of binding of the MCAM-Fcprotein to the cell line was determined by flow cytometry.

For the generation of rat antibodies capable of binding to human MCAM,hMCAM-Fc was generated by fusing the extracellular domain of human MCAMto human IgG and produced in CHO cells using standard techniques. Lou/Mrats were immunized with 250 μg of hMCAM-Fc protein in CFA (1:1 volume).Rats were boosted two times at two week intervals with hMCAM-Fc proteinin incomplete Freund's adjuvant (IFA) (1:1 volume). Hybridomas weregenerated from immunized rats using standard protocols and clones wereselected by Clonepix. CHO cells were transfected with the full lengthhuman MCAM gene and selected for stable expression using neomycin andstandard techniques. Parental CHO cells (MCAM negative) werefluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE)using standard techniques and mixed at a 1:1 ratio with unlabeled humanMCAM transfected CHO cells. Hybridoma supernatants were incubated withthis mixture of cells for 30 minutes and binding of potential human MCAMspecific antibodies was detected with a fluorescently labeled anti-ratsecondary antibody (Jackson Immuno) by flow cytometry.

For the generation of mouse antibodies capable of binding to human MCAM,hMCAM-Fc was generated by fusing the extracellular domain of human MCAMto human IgG and produced in CHO cells using standard techniques. Balb/cmice were immunized with 50 μg of hMCAM-Fc protein in CFA (1:1 volume).Mice were boosted two times at two week intervals with hMCAM-Fc proteinin incomplete Freund's adjuvant (IFA) (1:1 volume). Hybridomas weregenerated from immunized mice using standard protocols and clones wereselected by Clonepix. CHO cells were transfected with the full lengthhuman MCAM gene and selected for stable expression using neomycin andstandard techniques. Parental CHO cells (MCAM negative) werefluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE)using standard techniques and mixed at a 1:1 ratio with unlabeled humanMCAM transfected CHO cells. Hybridoma supernatants were incubated withthis mixture of cells for 30 minutes and binding of potential human MCAMspecific antibodies was detected with a fluorescently labeled anti-mousesecondary antibody (Jackson Immuno) by flow cytometry.

Supernatants from hybridomas that screened positive for human MCAMspecific antibodies were pre-incubated with fluorescently labeledhMCAM-Fc protein (5 μg/mL) for 30 minutes before addition to the lamininα4 expressing cell line WM2664 and neutralization of binding of thehMCAM-Fc protein to the cell line was determined by flow cytometry.

Nucleic Acid and Protein Manipulation

For microarray experiments, human CD4+ T cells were isolated as above,stained for CD161 and CCR6 (both from BD Pharmingen), and sorted intoCD4+/CD161−/CCR6− (non-TH17) and CD4+/CD161+/CCR6+ (TH17) cells fromthree independent healthy donors. RNA was isolated from half of thecells from each donor immediately (circulating) and the other half wasstimulated with plate bound anti-CD3 and soluble anti-CD28 as above, inthe absence of exogenous cytokines for four days (activated) before RNAisolation. RNA was amplified (Nugen) and hybridized on Human U133 Plus2.0 Array (Affymetrix). All microarray experiments were performed atExpression Analysis, Inc. (Durham, N.C.).

For determination of CDRs, total RNA was isolated from hybridoma cellsusing RNAquous-4PCR kit (Ambion), and was used for cDNA synthesis. Firstand second strand cDNA was synthesized using methods modified fromMarathon cDNA amplification (Clontech) with the cDNA adaptor ligated tothe 5′-end of the obtained dscDNA. The reverse specific primer wasdesigned based on the specific antibody isotype constant region sequencefor both heavy and light chains, and was used along with the adaptorprimer in the PCR amplification of both VL and VH fragments using PfuUltra DNA polymerase (Stratagene). The amplified PCR product was clonedinto pCR-Blunt-TOPO (Invitrogen), and the nucleotide sequence wasdetermined. Identical VL and VH sequences (those of clone 17) wereidentified from at least 3 out of 5 individual clones for both light andheavy chains.

For determination of IL-17 concentrations in the supernatant, ELISA wasperformed using a commercial kit (R&D Systems).

Fluorescence Microscopy/Standard Immunofluorescent Methods

Tissues from EAE induced mice were snap frozen in OCT and sectioned at10 μM. Sections were fixed in cold acetone and stained with directlyconjugated anti-pan-laminin (Novus Biologicals), MCAM-Fc, anti-CD31 (BDPharmingen), or anti-laminin α4 (Novus biological). In some experiments,MCAM-Fc was preincubated with anti-MCAM antibodies prior to addition totissues to ascertain neutralization of MCAM binding to its ligand ontissues.

Mouse Polarization Experiment

Splenocytes from mice immunized with PLP in CFA for 11 days wereisolated and cultured in the presence of PLP (5 μg/mL, HookeLaboratories). Where indicated, human TGFβ (5 ng/ml) and/or murine IL-23(20 ng/mL), and murine IL-1β (20 ng/mL) were added for five days in RPMIcontaining 10% heat-inactivated FCS (HyClone Laboratories), penicillin,streptomycin, L-glutamine, anti-IFNγ (5 μg/ml; R&D Systems), anti-IL4(0.5 μg/ml, R&D Systems) and β-ME (50 μM). All cytokines were from R&DSystems. Cells were stained with anti-CD4, anti-NK1.1 (both from BDPharmingen) and anti-MCAM generated as described above.

Example 1. MCAM is a Gene Up-Regulated in IL-17-Producing Human CD4+ TCells

To identify novel targetable molecules associated with TH17 cellinfiltration of the CNS, human CD4+ T cells from three healthy donorswere enriched by magnetic negative selection as described in Materialsand Methods above. After the enriched human CD4+ T cells were stainedfor surface expression of CD161 and CCR6, cells were FACS sorted intotwo populations: CCR6−/CD161− (representing circulating non-TH17 cells)and CCR6+/CD161+ (representing circulating TH17 cells) as described inMaterials and Methods above. RNA was isolated immediately from half ofthe cells in each population as described in Materials and Methodsabove. The other half was put into culture with plate-bound anti-CD3 andsoluble anti-CD28, without exogenous cytokines, for four days to obtainactivated non-TH17 cells and activated TH17 cells, respectively. RNA wassimilarly isolated from these two types of activated cells. RNA wassubject to microarray analysis as described in Materials and Methodsabove to identify genes specifically expressed in TH17 cells.

As shown in FIG. 1A, RORγt, a known TH17 transcription factor, wasup-regulated in both circulating and activated TH17 cells, while IL-17,as an activated TH17 marker, was nearly exclusively expressed in theactivated TH17 population. These results indicate that the aboveprocedures of separation and activation were successful. Microarrayanalysis identified MCAM as an up-regulated gene in both circulating andactivated TH17 cells—a profile similar to that of RORγt (FIG. 1A).

MCAM expressing T cells have been described previously as havingenriched expression among T cell clones generated from multiplesclerosis patients, and are particularly prominent at sites ofinflammation. See, e.g., Brucklacher-Waldert et al., Brian 132:3329-3341 (2009); see also Pickl et al., J. Immunol. 158: 2107-2115(1997). Here, the MCAM protein was found to be present on the surface ofa small population of CD4+ T cells (typically 3-5% of healthy donors).MCAM protein was also found to exist nearly entirely within the CD45RO+memory population of T cells (FIG. 1B). The human CD4+ T cells wereisolated as above, and stimulated for four hours with phorbol myristateacetate (PMA)/Ionomycin. The stimulated CD4+ T cells were analyzed forintracellular IL-17 and surface MCAM levels as described in Materialsand Methods above. As shown in FIG. 1C, although the majority of T cellsproducing IL-17 under these conditions were MCAM negative, MCAM proteinwas enriched on IL-17-producing cells. Only 2.3% of MCAM negative cells(2.18%/(2.18%+92.62%)) stained positive for IL-17; while 11.9% of MCAMexpressing cells (0.62%/(0.62%+4.58%)) were IL-17 positive. Given thesedata, MCAM is enriched in IL-17-producing human CD4+ T cells.

Furthermore, when CD4+/CD45RO+ memory T cells were separated intopurified populations of MCAM positive and MCAM negative cells andstimulated in vitro with anti-CD3 and anti-CD28, the MCAM positivepopulation produced nearly ten times as much IL-17 (data not shown). Themajority of the potential IL-17 production was found to be from thesmall population of T cells expressing MCAM. In one donor, only the MCAMpositive population produced detectable levels of IL-17. Thus, themajority of the potential IL-17 production is from the small populationof T cells expressing MCAM.

Example 2. MCAM Expressing T Cells are Effector Memory T Cells Having aUnique Integrin Expression Profile

The CD45RO+ memory population of human CD4 T cells can be segregatedinto (1) effector memory cells with tissue tropism, and (2) centralmemory cells with lymphoid tissue homing based upon expression of CCR7.See, e.g., Sallusto et al., Nature 401: 708-712 (1999).

To determine which subpopulation includes the MCAM expressing T cells,MCAM expression in T cells was further characterized by stainingperipheral human T cells with various markers (CCR6, CCR7, integrinsubunits alpha 4, beta 1, and beta 7) as described in Materials andMethods above. MCAM expressing CD4+ T cells were largely CCR7 negative,indicating that most are effector memory T cells, and would be morelikely to home to tissues (FIG. 2A). The TH17 enrichment protocolsuggested that MCAM expressing T cells obtained would bedisproportionately CCR6+. As shown in FIG. 2A, about 64% of MCAM+ cells(2.8%/(2.8%+1.6%)) express CCR6, while only 16.1% of MCAM negative cells(15.4%/(15.4%+80%)) express CCR6 (FIG. 2A). These data suggest that MCAMpositive cells would be largely tropic for areas where the ligand forCCR6, CCL20, is high. See, e.g., Liao et al., J. Immunol. 162: 186-194(1999).

The integrin expression pattern of MCAM expressing T cells was furthercharacterized. The majority of MCAM expressing T cells are integrin α4positive, but are largely integrin β7 negative and β1 positive (FIG.2B), which is a phenotype associated with the T cells involved in thepathogenesis of EAE (experimental autoimmune encephalomyelitis). See,e.g., Bauer et al., Proc. Nat'l Acad. Sci. USA 106: 1920-1925 (2009).

Example 3. MCAM Expressing T Cells are Expanded by IL1β and Produce theMajority of Both IL-17 and IL-22 Under TH17 Conditions

MCAM expressing CD4+ T cells, at only 3-5% of cells, is a small minorityof the T cell population. It is of interest to determine the conditionsunder which this population expands and exerts TH17 effector function.For this, human CD4+/CD45RO+ T cells were purified as described inMaterials and Methods above and stimulated in vitro with anti-CD3 andanti-CD28 in the presence of a number of cytokine conditions (TGFβ,IL-12, IL-1β, IL-23, and various combinations), and the percentage ofMCAM expressing cells, as well as IL-17 expressing cells, was determinedby flow cytometry (FIG. 3A). MCAM expression expanded upon stimulationwith IL-1β alone (16.4% in the absence of IL-1β vs. 38.1% in thepresence of IL-1β, FIG. 3B). Furthermore, while TGFβ alone did notexpand the MCAM positive population greatly, it functionedsynergistically with IL-1β, as the combination of both cytokinesresulted in more than half of the memory T cell population becoming MCAMpositive. Under the same conditions that expanded the population of MCAMexpressing cells, the population of IL-17 producing cells wasconcomitantly increased, with considerable enrichment within the MCAM+population under all cytokine conditions tested (FIG. 3C). In fact, inthe presence of TGFβ and IL-1β, more than 80% of the IL-17 producingcells (20.2%/(20.2%+4.4%)) were MCAM positive.

Additional to IL-17, the known TH17 associated cytokine IL-22 (Liang etal., J. Exp. Med. 203: 2271-2279 (2006)) was also elevated in MCAMexpressing T cells. IL-22 receptor is largely expressed on non-immunecells such as epithelial cells and functions in anti-microbial responsesas well as tissue remodeling. See, e.g., Dumoutier et al., J. Immunol.167: 3545-3549 (2001); see also Zenewicz et al., Int. Immunol. 23:159-163 (2011). Although IL-22 has been shown to be involved in bloodbrain barrier function, it is not absolutely required for induction orprogression of EAE. See, e.g., Kreymborg et al., J. Immunol. 179:8098-8104 (2007); see also Kebir et al., Nat. Med. 13: 1173-1175 (2007).In a similar fashion to IL-17, a significantly higher percentage ofMCAM+ cells expressed IL-22 (FIG. 3D).

TH17 cells have also been reported to express CCL20. See, e.g., Hirotaet al., J. Exp. Med. 204: 2803-2812 (2007). Similar to IL-17 and IL-22,there was a considerably higher population of MCAM expressing T cellsthat were positive for CCL20 (FIG. 3E), suggesting a possible positivefeedback loop in the migration of CCR6+ T cells.

While the above data are suggestive of a T cell population with aparticularly pathogenic phenotype, it was unexpected to observe thatMCAM expression was not mutually exclusive with intracellular FOXP3, andin fact, a slightly higher percentage of MCAM+ T cells were FOXP3positive (FIG. 3F). In the presence of increasing doses of TGFβ, thepercentage of MCAM+ cells that were FOXP3+ increased, while thepercentage of FOXP3 expressing cells in the MCAM− population remainedlargely unchanged. These results suggest that MCAM expressing cells havethe potential to function in an immunoregulatory role in the presence ofTGFβ.

Example 4. MCAM Binds to the ECM at Known Sites of T Cell Infiltrationof the CNS, and the MCAM Ligand is Laminin 411

The function of MCAM has been elucidated in tumor models, showing thatMCAM expression confers an adhesive, infiltrative, and ultimatelymetastatic phenotype to tumor cells. See, e.g., Xie et al., Cancer Res.57: 2295-2303 (1997). However, the ligand that MCAM binds remains to beidentified. Although the above data indicate that MCAM is enriched inTH17 cells, it is unknown whether MCAM is functionally involved in the Tcell infiltration of the CNS. It was thus of great interest to determine(1) where MCAM binds, i.e., the identity of the MCAM ligand, (2) whetherMCAM is critical to initial infiltration of TH17 cells into theuninflamed brain, and (3) whether the expression of the MCAM ligand isrequired at the established points of entry to the CNS.

An MCAM-Fc fusion protein was generated (as described in Materials andMethods above) to detect MCAM binding on healthy mouse tissue,particularly those regions known to be involved in T cell infiltration.As the choroid plexus has been suggested as a route of entry for TH17cells into the uninflamed brain, healthy choroid plexus tissue wasstained with MCAM-Fc and anti-laminin. As shown in FIGS. 4A and 4B, thechoroid plexus widely expresses the MCAM ligand, but is negative forMCAM. These results strongly suggest that (1) MCAM unlikely mediatesadhesion to the choroid plexus tissue through a homotypic MCAM/MCAMinteraction; and (2) there is an additional MCAM ligand withconsiderably more widespread expression than MCAM, whose expression waslimited to vascular endothelium within healthy tissues (FIG. 4C). It wasunexpected that MCAM-Fc bound nearly ubiquitously to healthy mousespinal cord (FIG. 4D) in a pattern that was suggestive of anextracellular matrix (ECM) protein, and specifically laminin. MCAM-Fcand anti-laminin co-localized on healthy mouse spinal cord (FIG. 4E),suggesting that the ligand for MCAM might be a form of laminin. MCAMligand was confirmed to be in the ECM, as it was exterior to theendothelial cell layer within the vasculature, as determined by CD31co-staining (FIG. 4F).

While MCAM co-localized with laminin within healthy mouse tissues, theidentity of the MCAM ligand was further confirmed by co-staining EAEtissues with laminin and MCAM-Fc. In regions of lymphocyte infiltration,it has been found that the basement membrane separates into two distinctmembranes, the endothelial basement membrane and the parenchymalbasement membrane with important distinctions in laminin isoformcomposition. See, e.g., Sixt et al., J. Cell Biol. 153: 933-945 (2001).When MCAM-Fc was used to stain the MCAM ligand within these regions, itwas found that MCAM-Fc stained only the endothelial basement membrane,while pan-laminin stained both the endothelia basement membrane and theparenchymal basement membrane (FIG. 4G). This same expression patternhas been noted for the laminin 411 (laminin 8 (α4β1γ1)). Co-localizationof MCAM-Fc protein and laminin alpha 4 was observed by using a lamininalpha 4 specific antibody (FIG. 4H), suggesting that laminin 411 is aligand for MCAM. Confirmation that a laminin molecule comprising analpha 4 chain is the ligand for MCAM, and which may contribute to theunique migratory capability of TH17 cells is described by Flanagan etal., PLoS ONE 7(7): e40443. doi:10.1371/journal.pone.0040443 (2012).

Example 5. Anti-MCAM Antibodies Block Binding of MCAM to Laminin 411

Monoclonal antibodies against mouse MCAM were generated as described inMaterials and Methods above. The specific binding between the monoclonalantibody and MCAM was confirmed by assessing the monoclonal antibody'sability to bind to cells transfected with either mouse or human MCAM.For this, untransfected cells were labeled with carboxyfluoresceinsuccinimidyl ester (CFSE) and mixed with unlabeled MCAM transfectedcells. Untransfected cells (in blue) could therefore be differentiated.As shown in FIG. 5A, clones 15 and 17 showed specific binding to mouseMCAM (top, orange) while only clone 17 bound to human MCAM (bottom,orange).

Next, the monoclonal antibodies were used to test their ability to blockthe binding of MCAM to its ligand. Murine or human MCAM-Fc protein (5μg/mL) was pre-incubated with isotype control antibody, clone 15, orclone 17 (10 μg/mL) for 30 minutes in PBS. The mixture was added tohealthy spinal cord tissue sections and subsequently characterized byfluorescence microscopy as described in Materials and Methods above.

As shown in FIG. 5B, both clones 15 and 17 could block binding of themurine MCAM-Fc protein to the tissue, while only clone 17 could blockhuman MCAM-Fc protein binding to the tissue. CDRs of clone 17 have beensequenced and are presented in FIGS. 6A (SEQ ID NO:2, light chain) and6B (SEQ ID NO:7, heavy chain). Non-denaturing Western blot analysisusing clone 17 on individual Fc domains of MCAM confirmed that clone 17binds specifically to a domain comprising amino acid residues 19 to 129of MCAM. This binding was confirmed by ForteBio analysis.

Furthermore, the MCAM monoclonal antibodies were shown to inhibit theinteraction between MCAM and its ligand, laminin 411. Parental CHO cells(CHOK1) or CHO cells transfected with mouse MCAM gene were preincubatedwith CHO culture media (DMEM), recombinant laminin 411 (10 μg/ml), orrecombinant laminin 511 (i.e., laminin 10 (α5β1γ1)) (10 μg/ml) at 37° C.for 45 minutes. Cells were washed, and specific binding of laminin 411,but not laminin 511, to MCAM was detected with a pan-laminin antibody byflow cytometry (FIG. 5C, top right panel). Preincubation of mouse MCAMtransfected CHO cells with the anti-MCAM antibody (clone 15 or clone 17,each at 20 μg/ml), prior to laminin incubation, abolished the binding ofMCAM to laminin 411 (FIG. 5C, bottom panels).

The above-presented data suggest that clone 17, which is capable ofspecifically blocking the binding of human MCAM to its ligand, might beuseful to prevent or treat various TH17-mediated diseases by inhibitingMCAM-mediated adhesion of TH17 cells to the vasculature and blocking themigration of TH17 cells into central nervous system.

Example 6. MCAM is Not Expressed on Circulating Mouse T Cells, but isInduced Following TH17 Polarization

Using the antibodies above, peripheral mouse blood was stained to detectMCAM expressing T cells in mice as described in Materials and Methodsabove. As previously described, mouse T cells lack expression of MCAM,while expression is noted on a population of NK cells (FIG. 7A). Theexpression of MCAM solely on memory T cells in humans suggests thatmice, if living in a clean environment with limited previous T cellactivation, would have to be polarized in order to generate a populationof MCAM expressing T cells. Considering the link between MCAM and TH17cells in humans, experiments were conducted to determine whether it waspossible to induce a population of MCAM expressing T cells in mice.Myelin proteolipid protein (PLP) specific T cells were generated byimmunizing wild type mice with PLP in the presence of complete Freund'sadjuvant (CFA) as described in Materials and Methods above. Splenocyteswere restimulated in vitro with 5 μg/mL PLP in the presence of theindicated cytokines and analyzed five days later for MCAM expression(FIG. 7B). In the absence of exogenous cytokines, the restimulation didnot induce statistically significant MCAM expression on CD4+ cells (ascompared to isotype control). In the presence of IL-23, a smallpopulation of MCAM expressing CD4+ T cells was detectable. While TGFβalone did not induce a sizable population of MCAM expressing T cells,the combination of TGFβ and IL-23 synergistically generated MCAMexpression among CD4+ T cells. Both of these cytokines have an importantrole in the polarization and effector function of mouse TH17 cells.Notably, MCAM was expressed on a population of CD4 high T cells whichhave been described to exclusively contain the pathogenic T cells inEAE. See, e.g., Li et al., J. Neuroimmunol. 192: 57-67 (2007). Thus,unlike humans, mice do not possess a population of circulating CD4+MCAM+T cells, but polarization under TH17 conditions with TGFβ and IL-23 issufficient to generate such a population. Mice remain a viable model tostudy the role of MCAM in the infiltration of CNS by pathogenic T cells.

Example 7. MCAM Blockade by an Anti-MCAM Antibody Inhibits EAE DiseaseProgression

EAE is a disease that is generated laboratory animals to producesymptoms similar to those of multiple sclerosis (MS) in humans. EAE isgenerally produced by injecting animals with different proteins from thecentral nervous system of other animals, for example, extracts of myelinbasic protein and whole spinal cord or brain tissue, or with T cellsthat specifically react to myelin. EAE is commonly used to follow thecourse the relapsing or progressive forms of MS. EAE has been served asa suitable animal model to both develop therapeutic agents for MS andstudy the specific disease processes of MS. See, e.g., Gold et al.,Brain 129: 1953-1971 (2006); see also Steinman et al., Ann. Neurol. 60:12-21 (2006).

The effects of MCAM blockade on disease progression were furtherexamined in a therapeutic model of EAE, wherein the TH17 polarizationoccurs in vivo (see Example 6). Mice were immunized with PLP 139-151peptide as described in Materials and Methods above. Immunized mice wererandomized into groups based on clinical scores and day of onset. On thesecond day following disease onset (EAE symptoms appeared between 12 and14 days post immunization), mice were treated (N=15 per group)intraperitoneally with either anti-MCAM antibody (clone 15) or isotypecontrol (Bioxcell) at 10 mg/kg body weight, and every day thereafter.Mice were monitored daily and scored for in a blinded manner (FIG. 8A),and body weights were obtained every 2-3 days (FIG. 8B). While MCAMblockade does not appear to affect the severity or duration of theongoing acute phase of the disease, relapse was delayed and wassignificantly less severe in mice treated with anti-MCAM antibody (clone15). These results are consistent with the idea that MCAM may not beessential for infiltration of immune cells during an existinginflammatory process, but may be involved in the subsequent recruitmentof antigen experienced pioneer T cells to initiate new inflammatorysites.

Example 8. Domain Binding Test for Murine Anti-MCAM Antibodies

The following protocol was used: ForteBio Domain Mapping Protocol.ForteBio anti-human IgG Fc biosensors were used to immobilize variousmouse MCAMhFc domains including full length mouse MCAMhFc protein on tobiosensor surface. These sensors were dipped into either clone 15 or 17MCAM specific antibody for detection of binding to these domains or fulllength protein. After loading these samples into a black 96 well plate,the Octet Red was programmed as follows: 60 seconds for baseline #1; 180seconds for loading various domains; 60 seconds for baseline #2; 180seconds for association of antibody to domain; and 240 seconds fordissociation of antibody from domain.

Reagents and Supplies Used:

-   -   1. Mouse MCAMhFc final concentration @ 5 ug/ml    -   2. Antibody clone 15 or 17 @ 5 ug/ml    -   3. ForteBio anti-human IgG Fc Capture (AHC) biosensors for        kinetics experiments, cat #18-5060    -   4. Block 96 well plate from Greiner Bio-one, cat #655209    -   5. ForteBio Octet Red machine    -   6. Fresh tissue culture medium, DMEM with 20% FCS, was used as        buffer for dilution

FIG. 10A demonstrates that clone 15 binds specifically to MCAM Fcdomains 1 and 2, but not Fc domain 1 alone. FIG. 10B demonstrates thatclone 17 binds specifically to either MCAM Fc domains 1 and 2, or Fcdomain 1 alone. For FIGS. 10A-B, clones 15 and 17 were tested againstthe following protein samples (all have human IgG Fc tag): Murine MCAM;Human Fc full length protein; Murine MCAM domain 1 (Ig1); Murine MCAMdomain 2 (Ig2); and Murine MCAM domain 1 and 2 (Ig1-2A).

Example 9. MCAM Domains Bind Laminin A4 (α4) Chain

The binding affinity of the human laminin-α4 to human MCAM IgG1-2A wasmeasured by Surface Plasmon Resonance on a Biacore T200 machine. HumanFc-specific F(ab′)₂ IgG (Jackson Laboratories) was immobilized on a CM5chip using amine coupling. The four flow cells of the CM5 chips dextransurface are activated by a 7 min injection of freshly prepared 1:1 50 mMNHS: EDC at a flow rate of 5 μl/min. 70 μl IgG solution (pH 4.5) wasinjected for 3 min to a density of up to 3 000 RU. The coupling is thenblocked by a 7 min injection of 1M ethanolamine to deactivate residualreactive sites. Recombinant human Fc-tagged MCAM IgG1-2A in degassed andfiltered HBS-P buffer containing 12 mg/ml BSA and 12 mg/mlcarboxy-methylated dextran sodium salt was captured by anti-Fc IgG to acapture level 1560 RU. Recombinant human Fc-tagged MCAM IgG1-2A wascentrifuged at 14 000 rpm for 5 min at 4° C. before injection for 20 minat a flow rate of 5 μl/min over the anti-Fc IgG containing surface. Flowcell 1 was left free of IgG to serve as a control surface. One flow cellwas used to capture recombinant human IgG1 Fc (R&D systems) to serve asa negative control. Recombinant human laminin-α4 (R&D systems) orrecombinant human laminin 411 (Biolamina) or recombinant human laminin511 (Biolamina) (negative control) was diluted in degassed and filteredHBS-P buffer containing 12 mg/ml BSA and 12 mg/ml carboxy-methylateddextran sodium salt to concentrations spanning 5-175 nM and injected (1min association, 3 min dissociation) over the MCAM IgG1-2A surfaces andcontrol surfaces at a flow rate of 10 μl/min. Buffer injections servedas negative control. Data evaluation: Data from the buffer injectionsand the control surface were subtracted to remove artifacts. The datawas fitted globally to a 1:1 interaction model using the Biaevaluationsoftware or Scrubber.

The laminin α-4 chain was found to bind specifically to the MCAM Fcdomains 1 and 2, but not to Fc domain 1 alone (data not shown). Thenegative controls included: a lack of binding of laminin 511 to eitherdomain, and a lack of binding of laminin 411 to hIgG1-Fc. Recombinanthuman laminin-α4 (R&D systems) binds to human Fc-tagged MCAM IgG1-2A(data not shown) at an affinity of 60 nM, but not to recombinant humanIgG1 Fc (R&D systems) (data not shown). Recombinant human laminin 411(Biolamina) binds to human Fc-tagged MCAM IgG1-2A at an affinity of 66nM as measured by steady state kinetics (data not shown) but not torecombinant human IgG1 Fc (R&D systems) (data not shown). The negativecontrol, recombinant human laminin 511 (Biolamina) does not bind tohuman Fc-tagged MCAM IgG1-2A (data not shown).

Example 10. Generation of New Anti-MCAM Monoclonal Antibodies

Mouse and rat monoclonal antibodies directed against human MCAM proteinwere generated as described in Materials and Methods above. The specificbinding between the monoclonal antibody and human MCAM was confirmed byassessing the monoclonal antibody's ability to bind to cells transfectedwith human MCAM. For this, untransfected cells were labeled withcarboxyfluorescein succinimidyl ester (CFSE) and mixed with unlabeledhuman MCAM transfected cells. Untransfected cells could, therefore, bedifferentiated.

Using these techniques, 823 independent mouse fusions clones wereisolated and shown to express an antibody capable of binding to humanMCAM. Additionally, 152 independent rat fusions clones were isolated andshown to express an antibody capable of binding to human MCAM.

Next, the anti-human MCAM monoclonal antibodies were used to test theirability to block the binding of human MCAM to its ligand. Human MCAM-Fcprotein (5 μg/mL) was pre-incubated with isotype control antibody, or 10μg/mL of the test monoclonal antibody for 30 minutes in PBS. The mixturewas added to healthy spinal cord tissue sections and subsequentlycharacterized by fluorescence microscopy as described in Materials andMethods above. Furthermore, parental CHO cells (CHOK1) or CHO cellstransfected with a human MCAM gene were preincubated with CHO culturemedia (DMEM), recombinant laminin 411 (10 μg/ml), or recombinant laminin511 (i.e., laminin 10 (α5β1γ1)) (10 μg/ml) at 37° C. for 45 minutes.Cells were washed, and specific binding of laminin 411, but not laminin511, to MCAM was detected with a pan-laminin antibody by flow cytometry.Preincubation of human MCAM transfected CHO cells with the anti-MCAMantibody (at 20 μg/ml), prior to laminin incubation, abolished thebinding of human MCAM to laminin 411.

Using this technique, it was shown that 87 of the 823 independent mousefusion clones and 26 of the 152 independent rat fusion clones describedabove expressed an antibody that was capable of blocking the interactionbetween human MCAM protein and its ligand, α-4 chain of laminin.

Example 11. Further Characterization of New Anti-MCAM MonoclonalAntibodies

The 87 independent mouse fusion clones and 26 independent rat fusionclones described in Example 10 above as being capable of (i) binding tohuman MCAM, and (ii) blocking the interaction between human MCAM and theα-4 chain of laminin were further characterized as follows. First, IC50quantitation for the ability of the monoclonal antibody to block thebinding of human MCAM to the α-4 chain of laminin was determined asfollows. CHO cells expressing human MCAM were incubated with ananti-human MCAM antibody (at various concentrations) for 30 minutes at 4degrees Celsius. Unbound antibody was then washed away, and the cellswere incubated with recombinant human laminin 411 at 20 ug/ml for 45minutes at 37 degrees Celsius. Unbound laminin was then washed away, andthe laminin bound to the surface of the cells was detected withfluorescently labeled anti-laminin antibodies. After washing, the amountof laminin bound to the surface was detected by flow cytometry, andIC50s were calculated based on the mean fluorescent intensity.

Using the above described assay, six independent anti-human MCAMmonoclonal antibody clones were identified as binding to human MCAM andhaving the greatest ability to block the interaction between human MCAMexpressed on the surface of cells and its binding ligand, human laminin411. These six anti-MCAM monoclonal antibody clones are herein referredto as (i) the mouse anti-human MCAM monoclonal clones 1174.1.3,1414.1.2, 1415.1.1, and 1749.1.3, and (ii) the rat anti-human MCAMmonoclonal antibody clones 2120.4.19 and 2107.4.10. Amino acid andnucleic acid sequences of the heavy and light chains of these antibodiesare provided in FIGS. 13-24. More specifically, in the above assay,IC50s for the monoclonal antibody clones 1174.1.3, 1414.1.2, 1415.1.1,1749.1.3, 2120.4.19, and 2107.4.10 were determined to be 0.469 ug/ml,0.431 ug/ml, 0.307 ug/ml, 0.545 ug/ml, 0.888 ug/ml, and 0.290 ug/ml,respectively. Moreover, experiments performed to determine the specificbinding affinity of each monoclonal antibody demonstrated that each wascapable of binding to human MCAM protein with high affinity (data notshown). As such, each of these specific monoclonal antibodies was verycapable of binding to human MCAM and inhibiting the interaction ofcell-expressed human MCAM with its α-4 laminin binding ligand. Incontrast, two control antibodies, a non-specific human IgG1 antibody anda previously described, fully human anti-MCAM antibody referred to asABX-MA1 (e.g., see Mills et al., Cancer Res. 62:5106 (2002), and U.S.Pat. Nos. 6,924,360, 7,067,131, and 7,090,844) were both incapable ofblocking the binding interaction between human MCAM and its laminin 411counterpart. As such, the six specific monoclonal antibodies identifiedabove possess the novel ability to both (i) bind with high affinity tohuman MCAM on the surface of living cells, and (ii) block theinteraction of cell expressed human MCAM with a laminin proteincomprising an α-4 laminin polypeptide chain.

Example 12. Domain Binding Analysis for New Anti-MCAM MonoclonalAntibodies

The technique described in Example 8 above was employed to determine thelocation of the antigen epitope on the human MCAM protein that isrecognized and bound by monoclonal antibody clones 1174.1.3, 1414.1.2,1415.1.1, 1749.1.3, 2120.4.19, and 2107.4.10. The results from theseanalyses are as follows.

Monoclonal antibody clones 1174.1.3, 1414.1.2, 1415.1.1, and 1749.1.3were all shown to bind to an antigenic epitope found on domain 3 of thehuman MCAM protein, defined specifically by amino acids 244-321 (SEQ IDNO:24) of the human MCAM protein. These monoclonal antibodies were notcapable of binding to human MCAM domain 1 (namely amino acids 19-129,SEQ ID NO:22), domain 2 (namely amino acids 139-242, SEQ ID NO:23), orthe combination of domains 1 and 2 (namely, amino acids 19-242). Hence,monoclonal antibody clones 1174.1.3, 1414.1.2, 1415.1.1, and 1749.1.3define a novel antigenic epitope located within domain 3 of the humanMCAM protein.

Monoclonal antibody clones 2120.4.19, and 2107.4.10 were each shown tobind to an antigenic epitope defined by the combination of human MCAMdomains 1 (namely amino acids 19-129, SEQ ID NO:22), and domain 2(namely amino acids 139-242, SEQ ID NO:23). Neither of these twomonoclonal antibodies bound to human MCAM domain 1 by itself. Hence,monoclonal antibody clones 2120.4.19, and 2107.4.10 define a novelantigenic epitope determined by the presence of both human MCAM proteindomains 1 and 2.

In contrast to the above, the previously described fully human anti-MCAMantibody ABX-MA1 binds to a different antigenic epitope than thosedescribed above, namely an antigenic epitope that is fully defined andencompassed within human MCAM domain 1 only.

Given these results, since each of monoclonal antibody clones 1174.1.3,1414.1.2, 1415.1.1, 1749.1.3, 2120.4.19, and 2107.4.10 are capable ofboth (i) binding to human MCAM, and (ii) blocking the interactionbetween human MCAM and an α-4 laminin containing protein, whereas theABX-MA1 antibody is capable of only binding to human MCAM, but notblocking the interaction between human MCAM and an α-4 laminincontaining protein, these results demonstrate that human MCAM domain 2,human MCAM domain 3, and the combination thereof play a role in thebinding interaction with α-4 laminin chain. Given this, it is clear thatantibodies which bind to human MCAM domain 2, human MCAM domain 3,and/or the combination thereof would find use as agents capable ofblocking the interaction between human MCAM and α-4 laminin and,thereby, find use for inhibiting the various consequences describedherein resulting from that interaction. In contrast, antibodies thatbind to an antigenic epitope defined solely by human MCAM domain 1 (suchas the ABX-MA1 antibody described herein) are not useful for blockingthe MCAM/α-4 laminin interaction and its various downstream biologicalconsequences.

Example 13. Generation of Humanized Anti-MCAM Antibodies

Various humanized anti-MCAM antibodies were generated according to thefollowing protocol. First, a three-dimensional molecular model of thevariable regions was constructed using JN Biosciences' proprietaryalgorithm. Second, the framework amino acid residues important for theformation of the CDR structure or necessary for the binding to antigenwere identified using the molecular model. In parallel, cDNA-derivedhuman VH and VL amino acid sequences with high homology to the VH and VLamino acid sequences, respectively, were selected. Lastly, CDR sequencestogether with framework amino acid residues important for CDR structureor antigen binding were grafted from VH and VL into the correspondingselected human framework sequences.

FIGS. 25-27 depict the alignment of various 1749, 2107, and 2120 heavyand light chain sequences. Residue numbering is according to Kabatnumbering. Different mutations to the framework (FR) amino acid residuesinvolved in CDR formation and antigen binding were identified dependingupon the version of antibody. For example, exemplary mutations of the1749 antibodies are depicted in FIG. 25A (boxed residue between CDR-H2and CDR-H3 (A93T) affects CDR contact; the A93T mutation combined withmutations of the boxed residue between CDR-H1 and CDR-H2 (G44R) affectsVH/VL interface and CDR conformation) and FIG. 25B (Mutations of theboxed residue between CDR-L2 and CDR-L3 (S63T) affects CDR2 and antigencontact). Exemplary mutations of the 2107 antibodies are depicted inFIG. 26A (boxed residues before CDR-H1 (S30T), between CDR-H1 and CDR-H2(I37V and L48I), and between CDR-H2 and CDR-H3 (K71R) affect CDRcontacts; and S30T, I37V, L48I and K71R mutations combined with amutation of the boxed residue immediately after CDR-H2 (T68S) affectantigen/antibody interactions); and FIG. 26B (boxed residues betweenCDR-L1 and CDR-L2 (Y36F), and between CDR-L2 and CDR-L3 (V58I) affectCDR contact; and Y36F and V58I mutations combined with (i) an additionalmutation of the boxed residue between CDR-L1 and CDR-L2 (Q38L) affectCDR structure; or (ii) an additional mutation before CDR-L1 (T22N)affect antibody antigen interaction). Exemplary mutations of the 2120antibodies are depicted in FIG. 27A (boxed residues in CDR-H1 (S30T),between CDR-H1 and CDR-H2 (I37V and L48I), and between CDR-H2 and CDR-H3(K71R) affect CDR contact; and S30T, I37V, L48I, and K71R mutationscombined with an additional mutation after CDR-H2 (T68S) affect CDRcontact); and FIG. 27B (boxed residues between CDR-L1 and CDR-L2 (L46Vand Y49F) and between CDR-L2 and CDR-L3 (V58I) affect CDR contact; boxedresidues between CDR-L1 and CDR-L2 (L46V and Y49F) affect CDR contact;and L46V, Y49F, and V58I mutations combined with an additional mutationbefore CDR-L1 (T22N) affect antibody/antigen interaction).

Several versions of each chain were designed (standard vs. aggressive orconservative). For those antibodies that contained N-deamidation motifs(NG), mutations to the asparagines or glycine were introduced into thestandard version. The various humanized V regions were synthesized witha heterologous signal sequence and cloned into expression vectorscontaining human CK (VL) or human IgG1 (VH).

The heavy and light chain plasmids were co-transfected into 293F cellswith the FreeStyle™ MAX transfection regent (Invitrogen) according tothe manufacturer's protocol. The expressed antibody was purified withprotein A PhyTip columns (Phynexus) and quantified via OD280.

The apparent affinities of the humanized antibodies were compared to theparental rodent or chimeric antibody in a competitive ELISA according tothe following protocol.

ELISA plates were coated with recombinant hMCAM-His, and blocked withcasein buffer to prevent non-specific binding. Biotinylated rodent orchimeric antibody was added at a subsaturating concentration, in thepresence or absence of 3× increasing concentrations of unlabeledcompetitor (humanized antibody, rodent, or chimeric). After washing toremove unbound antibody, streptavidin HRP was added to allow detectionof the biotinylated antibody. The ELISA was developed with TMB substrateand the OD450 was measured. The IC50 of the unlabeled competitor wasdetermined using the GraphPad Prism5 software.

Table 2 summarizes the design of humanized sequences (see also FIGS.25-27 and 30-32).

TABLE 2 Donor Framework Mutations 1749 VH1 U96282 IGHV3-7*02 A93T VH2U96282 IGHV3-7*02 VH1 + G44R VL1 X02990 IGKV4-1*01 None VL2 X02990IGHKV-1*01 VL1 + S63T 2107 VH1 AF062133 IGHV2-26*01 S30T*, I37V, L48Iand K71R VH2 AF062133 IGHV2-26*01 VH1 mutations + T68S VH3 AF062133IGHV2-26*01 VH1 mutations + D72N VH4 AF062133 IGHV2-26*01 VH1mutations + N32S VH5 AF062133 IGHV2-26*01 VH1 mutations + N32Q VH6AF062133 IGHV2-26*01 VH1 mutations + G33A VL1 U86803 IGKV1-27*01 Y36F,V58I VL2 U86803 IGKV1-27*01 VL1 mutations + Q38L VL3 U86803 IGKV1-27*01VL1 mutations + T22N 2120 VH1 AF062133 IGHV2-26*01 S30T*, I37V, L48I andK71R VH2 AF062133 IGHV2-26*01 VH1 mutations + T68S VH3 AF062133IGHV2-26*01 VH1 mutations + N32S VH4 AF062133 IGHV2-26*01 VH1mutations + N32Q VH5 AF062133 IGHV2-26*01 VH1 mutations + G33A VL1X84343 IGKV1-39*01 L46V, Y49F and V58I VL2 X84343 IGKV1-39*01 L46V, Y49FVL3 X84343 IGKV1-39*01 VL1 + T22N

The heavy and light chain plasmids were co-transfected into 293F cellswith the FreeStyle™ MAX transfection regent (Invitrogen) according tothe manufacturer's protocol. The expressed antibody was purified withprotein A PhyTip columns (Phynexus) and quantified via OD280.

The apparent affinities of the humanized antibodies were compared to theparental rodent or chimeric antibody in a competitive ELISA according tothe following protocol:

ELISA plates were coated with recombinant hMCAM-His, and blocked withcasein buffer to prevent non-specific binding. Biotinylated rodent orchimeric antibody was added at a subsaturating concentration, in thepresence or absence of 3× increasing concentrations of unlabeledcompetitor (humanized antibody, rodent, or chimeric). After washing toremove unbound antibody, streptavidin HRP was added to allow detectionof the biotinylated antibody. The ELISA was developed with TMB substrateand the OD450 was measured. The IC50 of the unlabeled competitor wasdetermined using the GraphPad Prism5 software.

The affinities were measured using the ForteBio Octet Red. Anti-human Fcsensors were used to capture the humanized antibodies, and severalconcentrations of hMCAMHis analyte were used to determine the affinityusing a 1:1 fitting model.

The potencies of the antibodies were measured in the laminin/FACS assayaccording to the following protocol: recombinant laminin 411(Biolaminate) was added to hMCAM expressing CHO cells in the presence orabsence of varying concentrations of the humanized, rodent, or chimericantibodies. Following incubation for 30-45 minutes, the cells werewashed and anti-laminin conjugated to AF650 (NovusBio) was added todetect the bound laminin. The cells were run on a flow cytometer tomeasure the laminin binding signal.

Table 3 provides the constructs used for transfection.

TABLE 3 Construct Description h1749VH1 Standard h1749VH2 Conservativeh1749VL1 Standard h1749VL2 Conservative h2120_VH1 Standard h2120_VH2Conservative h2120_VH3 Standard + N-S h2120_VH4 Standard + N-Q h2120_VH5Standard + G-A h2120_VL1 Standard h2120_VL2 Aggressive h2120_VL3Conservative h2107_VH1 Standard h2107_VH2 Conservative h2107_VH3Standard + glyc h2107_VH4 Standard + N-S h2107_VH5 Standard + N-Qh2107_VH6 Standard + G-A h2107_VL1 Standard h2107_VL2 Conservative--rech2107_VL3 Conservative

Table 4 describes the specific transfection experiments.

TABLE 4 Transfection--round 1 h1749VH1 + h1749VL1 Standard VH and VLh1749VH2 + h1749VL2 Conservative VH and VL h2120_VH1 + h2120_VL3Standard VH + conservative VL h2120_VH2 + h2120_VL3 Conservative VH +conservative VL h2120_VH3 + h2120_VL3 N-S deamidate VH + conservative VLh2107_VH1 + h2107_VL2 Standard VH + conservative VL h2107_VH2 +h2107_VL2 Conservative VH + conservative VL h2107_VH3 + h2107_VL2Standard VH/restore N-gly + conservative VL h2107_VH4 + h2107_VL2 N-Sdeamidate mut VH + conservative VL Transfection--round 2 h1749VH1 +h1749VL2 Standard VH and conservative VL h1749VH2 + h1749VL1Conservative VH and standard VL h2120_VH4 + h2120_VL3 N-Q deamidate VH +conservative VL h2120_VH5 + h2120_VL3 G-A deamidate VH + conservative VLh2120_VH1 + h2120_VL1 Standard VH + standard VL h2120_VH1 + h2120_VL2Standard VH + aggressive VL h2107_VH1 + h2107_VL1 Standard VH andstandard VL h2107_VH1 + h2107_VL3 Standard VH and other VL h2107_VH5 +h2107_VL2 N-Q deamidate VH + conservative VL h2107_VH6 + h2107_VL2 G-Adeamidate VH + conservative VL h2107_VH5 + h2107_VL3 N-Q deamidate VH +other VL h2107_VH6 + h2107_VL3 G-A deamidate VH + other VL h2107_VH5 +h2107_VL1 N-Q deamidate VH + standard VL h2107_VH6 + h2107_VL1 G-Adeamidate VH + standard VL

Table 5 shows the relative affinities of the humanized antibodiescompared to the rodent parent as measured by ForteBio and competitiveELISA, as well as the expression levels for the first round oftransfections. FIG. 28A-C compares the blocking of various 1749, 2120,and 2107 antibodies of MCAM binding to laminin from the first round oftransfections.

TABLE 5 Forte ELISA Expt. #1 Expt. #2 Expt. #1 Expt. #2 Fold over Foldover Fold over Fold over Expression Transfection--round 1 rodent rodentrodent rodent level rodent 1749 1.00 1.00 1.00 1.00 h1749VH1 + h1749VL12.50 2.41 1.26 1.35 7.2 mg/L h1749VH2 + h1749VL2 0.73 1.09 1.28 1.46 7.2mg/L chimeric 1749 0.79 0.81 0.97 chimeric 1749 TM 1.07 rodent 2120 1.001.00 1.00 1.00 h2120_VH1 + h2120_VL3 5.64 6.21 2.23 2.42 22 mg/Lh2120_VH2 + h2120_VL3 6.57 6.43 1.93 2.62 16 mg/L h2120_VH3 + h2120_VL316.14 3.47 22 mg/L chimeric 2120 0.97 1.72 rodent 2107 1.00 1.00 1.001.00 h2107_VH1 + h2107_VL2 2.37 3.40 1.29 1.32 12 mg/L h2107_VH2 +h2107_VL2 2.54 3.58 1.32 1.48 26.7 mg/L   h2107_VH3 + h2107_VL2 2.541.62 14.6 mg/L   h2107_VH4 + h2107_VL2 5.59 11.72 26.7 mg/L   chimeric2107 0.68 1.01 chimeric 2107 TM 0.96

Table 6 shows the measured affinity by ForteBio, competitive ELISA, andfunctional blocking data (laminin/FACS assay) compared to the rodentparent, as well as the expression levels, from the second round oftransfections.

TABLE 6 Blocking ELISA Expt#1 Expt#2 Forte Forte Fold fold fold Foldover Fold over over over over Expression Transfection--round 2 rodentrodent rodent rodent rodent level h1749VH1 + h1749VL2 2.5 1.0 1.1 1.26.9 mg/L  h1749VH2 + h1749VL1 1.2 1.0 1.5 1.8 3.2 mg/L  h1749VH1 +h1749VL1 2.5 1.0 1.4 1.4 7.2 mg/L  h1749VH2 + h1749VL2 0.7 1.1 1.8 1.77.2 mg/L  chimeric 1749 0.6 1.4 1.4 rodent 1749 1.0 1.0 1.0 1.0h2120_VH4 + h2120_VL3 17.4 5.0 3.8 5.6 15 mgL h2120 _(—) VH5 + h2120_(—) VL3 1.1 1.2 2.4 1.2 1.5 22 mg/L h2120_VH1 + h2120_VL1 8.8 3.1 2.03.5 17 mg/L h2120_VH1 + h2120_VL2 10.8 3.1 4.6 12.6  2 mg/L h2120_VH1 +h2120_VL3 5.9 5.8 1.8 1.7 2.8 22 mg/L rodent 2120 1.0 1.0 1.0 1.0 1.0h2107_VH1 + h2107_VL1 12.9 2.1 2.3 2.1 25 mg/L h2107_VH1 + h2107_VL316.3 2.3 2.6 2.0 22 mg/L h2107 _(—) VH5 + h2107 _(—) VL2 5.9 6.0 1.3 1.61.5 19 mg/L h2107_VH6 + h2107_VL2 5.2 5.5 3.5 4.8 2.3  3 mg/Lh2107_VH5 + h2107_VL3 33.2 3.4 19 mg/L h2107_VH6 + h2107_VL3 22.1 9.9  3mg/L h2107_VH5 + h2107_VL1 28.2 3.7 18 mg/L h2107_VH6 + h2107_VL1 36.815.3  3 mg/L h2107_VH1 + h2107_VL2 3.0 2.8 1.4 1.9 1.4 12 mg/L h2107_(—) VH2 + h2107 _(—) VL2 2.7 2.9 1.3 1.4 1.5 26.7 mg/L   rodent 21071.0 1.0 1.0 1.0 chimeric 2107 1.0

Overall, the data demonstrates that the various 1749 humanizedantibodies have <2× reduction in potency and affinity compared to therodent. The various 2120 humanized antibodies have a >5× reduction inaffinity as measured by ForteBio, and most have a >2-3× reduction inapparent affinity and potency as measured by the competitive ELISA andlaminin blocking assay, with the exception of VH5VL3 (G-A N-deamidationmutant VH/conservative VL), which had a <2× reduction in affinity andpotency. Many of the 2107 humanized antibodies have a significant lossof affinity and potency, with the exception of VH1VL2, VH2VL2, andVH3VL2 (conservative light chain with standard, conservative, and therestored N-glycosylation site D72N heavy chains); these generally had<3× reduction and affinity and potency while retaining the N-deamidationsite.

Certain candidate antibodies were re-expressed and tested for theiraffinity by ForteBio and their IC₅₀. The results are provided in Table 7below and in FIG. 29.

TABLE 7 Blocking Forte kD IC50 Expression h2120VH5VL3 1.3 0.7 12.7 mg/Lh2107VH2VL2 1.4 0.8   20 mg/L h1749VH2VL1 0.67 0.4  3.3 mg/L

Example 14. Analysis of MCAM Blockade by a Humanized Anti-MCAM Antibodyand Inhibition of EAE Disease Progression

The humanized antibodies described herein, e.g., Example 13, are testedfor their effect on EAE disease progression according to the protocolprovided in Example 7.

Example 15. Use of Humanized Antibodies to Analyze MCAM ExpressionFollowing TH17 Polarization

The humanized antibodies described herein, e.g., Example 13, are used toanalyze MCAM expression following TH17 polarization according to theprotocol provided in Example 6.

Example 16. DNFB-Induced Skin Inflammation in Mice

DNFB (2,4-dinitrofluorobezene, Sigma) was diluted in acetone and oliveoil (4/1). Abdomen of BALB/c mice (6-10 weeks old) were shaved andsensitized with 25 ul of 0.5% DNFB solution on days 0 and 1 as a modelof allergic contact dermatitis and psoriasis. Animals receivedintraperitoneal treatment of antibodies (Anti-MCAM Clone 15 and Isotype,10 mg/kg) on days 6 and 7. Right mouse ear was challenge with 5 ul of0.2% DNFB and the left ear received vehicle (acetone/olive oil) on day 7(Nakae et al., Immunity. 2002 September; 17(3):375-871). Mice wereeuthanized on day 8 and ear thickness or swelling was monitored using amicrometer (Mitutoyo, USA). The ear swelling was calculated as[(T1+T2+T3+T4)/4 left ear]−[(T1+T2+T3+T4)/4 right ear], where Trepresent four different values of ear thickness. FIG. 30 shows theantibody significantly inhibited inflammation.

Example 17. Inhibition of Tumor Growth Nude SCID Mice

5×10⁵ human MCAM expressing melanoma cells (WM2664) were injectedsubcutaneously into male nude severe combined immunodeficiency (SCID)mice. Mice were treated weekly with 1 mg of total antibody (0.5 mg ofeach antibody in the combination group) weekly beginning on day 4 posttumor implantation. The antibodies used were anti-mouse MCAM clone 15 oranti-human MCAM clone 2120.4.19 or a combination thereof. Tumors weremeasured in a blinded fashion with calipers 2× per week and tumor volumewas determined by standard formula (Volume=(width)2×length/2) (FIG.31A). On day 40 post tumor implantation, mice were euthanized, andtumors were removed intact, and weighed (FIG. 31B). Each antibodyinhibited tumor growth and inhibition was most notable in thecombination treatment. It is believed the combination treatment is moreefficient in mice containing human cancer cells. The human cancer cellsexpress human MCAM and human laminin, and the mice express mouse MCAMand mouse laminin Because mouse and human MCAM/laminin bind to eachother, antibodies with specificity for human and mouse MCAM actsynergistically to inhibit tumor growth. In a human, where all MCAM andlaminin are human, human anti-MCAM clone 2120.4.19 (or similar antibody)is expected to be similarly effective as the combination treatment inmice.

What is claimed is:
 1. An isolated anti-MCAM antibody, or antigen binding fragment thereof, said antibody or antigen binding fragment thereof comprising three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3), wherein: (a) HVR-L1 comprises the amino acid sequence of SEQ ID NO:61, HVR-L2 comprises the amino acid sequence of SEQ ID NO:62, HVR-L3 comprises the amino acid sequence of SEQ ID NO:63, HVR-H1 comprises the amino acid sequence of SEQ ID NO:66, HVR-H2 comprises the amino acid sequence of SEQ ID NO:67, and HVR-H3 comprises the amino acid sequence of SEQ ID NO:68; (b) HVR-L1 comprises the amino acid sequence of SEQ ID NO:41, HVR-L2 comprises the amino acid sequence of SEQ ID NO:42, HVR-L3 comprises the amino acid sequence of SEQ ID NO:43, HVR-H1 comprises the amino acid sequence of SEQ ID NO:46, HVR-H2 comprises the amino acid sequence of SEQ ID NO:47, and HVR-H3 comprises the amino acid sequence of SEQ ID NO:48; (c) HVR-L1 comprises the amino acid sequence of SEQ ID NO:51, HVR-L2 comprises the amino acid sequence of SEQ ID NO:52, HVR-L3 comprises the amino acid sequence of SEQ ID NO:53, HVR-H1 comprises the amino acid sequence of SEQ ID NO:56, HVR-H2 comprises the amino acid sequence of SEQ ID NO:57, and HVR-H3 comprises the amino acid sequence of SEQ ID NO:58; (d) HVR-L1 comprises the amino acid sequence of SEQ ID NO:31, HVR-L2 comprises the amino acid sequence of SEQ ID NO:32, HVR-L3 comprises the amino acid sequence of SEQ ID NO:33, HVR-H1 comprises the amino acid sequence of SEQ ID NO:36, HVR-H2 comprises the amino acid sequence of SEQ ID NO:37, and HVR-H3 comprises the amino acid sequence of SEQ ID NO:38; or (e) HVR-L1 comprises the amino acid sequence of SEQ ID NO:85, HVR-L2 comprises the amino acid sequence of SEQ ID NO:86, HVR-L3 comprises the amino acid sequence of SEQ ID NO:87, HVR-H1 comprises the amino acid sequence of SEQ ID NO:90, HVR-H2 comprises the amino acid sequence of SEQ ID NO:91, and HVR-H3 comprises the amino acid sequence of SEQ ID NO:92.
 2. The isolated antibody of claim 1 which is a chimeric or humanized antibody.
 3. The isolated antibody of claim 1 which is an IgG1 antibody.
 4. The isolated antibody of claim 1 which is produced in bacteria or CHO cells.
 5. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein: (a) the light chain variable region comprises the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:99; and (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:93, SEQ ID NO:94 or SEQ ID NO:95.
 6. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:98 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:94.
 7. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:98 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95.
 8. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:99 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:94.
 9. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:99 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95.
 10. The isolated antibody of claim 5 which is a chimeric or humanized antibody.
 11. The isolated antibody of claim 5 which is an IgG1 antibody.
 12. The isolated antibody of claim 5 which is produced in bacteria or CHO cells.
 13. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:97 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:93.
 14. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein: (a) the light chain variable region comprises the amino acid sequence of SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112; and (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105 or SEQ ID NO:
 106. 15. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 14, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:109 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:101.
 16. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 14, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:111 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:102.
 17. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 14, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:111 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:103.
 18. The isolated anti-MCAM antibody, or antigen binding fragment thereof, of claim 14, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:111 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:104.
 19. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein: (a) the light chain variable region comprises the amino acid sequence of SEQ ID NO:30; and (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35.
 20. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein: (a) the light chain variable region comprises the amino acid sequence of SEQ ID NO:40; and (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:45.
 21. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein: (a) the light chain variable region comprises the amino acid sequence of SEQ ID NO:50; and (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:55.
 22. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has at least 95% sequence identity to SEQ ID NO:111 with any variation from SEQ ID NO:111 occurring outside the hypervariable regions of the light chain variable region; and the heavy chain variable region has at least 95% sequence identity to SEQ ID NO:103 with any variation from SEQ ID NO:103 occurring outside the hypervariable regions of the heavy chain variable region.
 23. The isolated anti-MCAM antibody, or antigen binding fragment thereof of claim 1, said antibody or antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region has at least 95% sequence identity to SEQ ID NO: 95 with any variation from SEQ ID NO:95 occurring outside the hypervariable regions of the light chain variable region and the heavy chain variable region has at least 95% sequence identity to SEQ ID NO:98 with any variation occurring outside the hypervariable regions of the heavy chain variable region.
 24. A pharmaceutical composition comprising an antibody, or antigen binding fragment thereof, according to any of claim 1, 5, 6, 7, 8, 9, 14, 16, 17, or
 18. 25. An article of manufacture comprising the pharmaceutical composition of claim
 24. 